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WO2020233650A1 - 核酸、药物组合物与缀合物及制备方法和用途 - Google Patents

核酸、药物组合物与缀合物及制备方法和用途 Download PDF

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WO2020233650A1
WO2020233650A1 PCT/CN2020/091484 CN2020091484W WO2020233650A1 WO 2020233650 A1 WO2020233650 A1 WO 2020233650A1 CN 2020091484 W CN2020091484 W CN 2020091484W WO 2020233650 A1 WO2020233650 A1 WO 2020233650A1
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nucleotide sequence
nucleotide
seq
sirna
nucleotides
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PCT/CN2020/091484
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English (en)
French (fr)
Inventor
张鸿雁
高山
康代武
刘涛
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苏州瑞博生物技术股份有限公司
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Priority to CN202080007282.1A priority Critical patent/CN113227376B/zh
Priority to JP2021569111A priority patent/JP2022533722A/ja
Priority to CA3139195A priority patent/CA3139195A1/en
Priority to AU2020280438A priority patent/AU2020280438A1/en
Priority to CN202410323310.0A priority patent/CN118256498A/zh
Priority to EP20810327.5A priority patent/EP3974532A4/en
Priority to US17/612,912 priority patent/US20230193277A1/en
Priority to KR1020217041711A priority patent/KR20220011689A/ko
Publication of WO2020233650A1 publication Critical patent/WO2020233650A1/zh

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Definitions

  • the present disclosure relates to a nucleic acid capable of inhibiting plasma coagulation factor XI (Factor XI, FXI) gene expression 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.
  • Plasma coagulation factor XI (Factor XI, hereinafter referred to as FXI) is a key component of the contact activation pathway, which contributes to the production of thrombin, which is involved in fibrin formation and fibrinolysis An important component of protection.
  • High FXI levels are a risk factor for venous thrombosis.
  • Small interfering RNA can be based on the mechanism of RNA interference (RNAi) to inhibit or block the expression of any target gene of interest in a sequence-specific manner, thereby achieving the purpose of treating diseases.
  • RNAi RNA interference
  • siRNAs and their modified sequences provided in the present disclosure can specifically inhibit the expression of FXI gene, and that pharmaceutical compositions or siRNA conjugates containing siRNA can specifically target the liver, thereby Inhibiting the expression of FXI gene in the liver to realize the treatment or prevention of thrombotic diseases, thus completing the present invention.
  • the present disclosure provides the first siRNA capable of inhibiting FXI gene expression.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified.
  • Z 1 is U
  • Z 2 is A
  • 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.
  • the present disclosure provides a second siRNA capable of inhibiting FXI gene expression.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified.
  • Z 5 is U
  • Z 6 is A
  • 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 present disclosure provides a third siRNA capable of inhibiting FXI gene expression.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified.
  • 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 present disclosure provides a fourth siRNA capable of inhibiting FXI gene expression.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified.
  • Z 13 is U
  • Z 14 is A
  • 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 present disclosure provides a fifth siRNA capable of inhibiting FXI gene expression.
  • 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 and the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary A double-stranded region is formed, wherein the 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 nucleotide sequence shown in SEQ ID NO: 242 has the same length and no more than 3 nucleotide differences:
  • Z 17 is U
  • Z 18 is A
  • 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.
  • the present disclosure provides a sixth siRNA capable of inhibiting FXI gene expression.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified.
  • Z 21 is G
  • Z 22 is C
  • 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.
  • the present disclosure provides a seventh siRNA capable of inhibiting FXI gene expression.
  • 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 and the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary A double-stranded region is formed, wherein the 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 nucleotide sequence shown in SEQ ID NO: 362 has the same length and 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.
  • the present disclosure provides an eighth siRNA capable of inhibiting FXI gene expression.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified.
  • Z 29 is U
  • Z 30 is A
  • 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 ninth siRNA capable of inhibiting FXI gene expression.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified.
  • Z 33 is A
  • Z 34 is U
  • the nucleotide sequence I contains the nucleotide Z 35 whose position corresponds to Z 33
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 34 Acid Z 36
  • the Z 36 is the first nucleotide at the 5'end of the antisense strand.
  • the present disclosure provides a pharmaceutical composition that contains the siRNA of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure provides an siRNA conjugate that contains 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 thrombotic diseases and/or thrombotic diseases caused by abnormal expression of the FXI gene. Or the use of drugs for ischemic stroke.
  • the present disclosure provides a method for treating and/or preventing thrombotic diseases and/or ischemic stroke, the method comprising adding an effective amount of the siRNA and/or pharmaceutical composition of the present disclosure and/or Or the siRNA conjugate is administered to subjects suffering from thrombotic diseases and/or ischemic stroke.
  • the present disclosure provides a method for inhibiting FXI gene expression in liver cells, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver. Cell contact.
  • the present disclosure provides a kit containing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
  • siRNA, pharmaceutical composition and siRNA conjugate provided in the present disclosure have good stability, high FXI mRNA inhibitory activity, low off-target effect, and/or can significantly treat or alleviate thrombotic diseases and/or ischemia Symptoms of sexual stroke.
  • the siRNA, pharmaceutical composition or siRNA conjugate provided in the present disclosure shows excellent target gene inhibitory activity in in vitro cell experiments. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% in liver cells Or 95% inhibition rate of target gene expression. In some embodiments, the present disclosure provides siRNA suppressed psiCHECK system of FXI mRNA of the IC 50 activity of FXI mRNA between the IC 50 0.013-0.119nM. In some embodiments, the siRNA provided in the present disclosure shows high inhibitory activity in HepG2 cells, with an IC 50 for FXI mRNA between 1.49-11.1 nM.
  • the siRNA conjugate provided in the present disclosure shows high inhibitory activity in primary mouse liver cells, with an IC 50 for FXI mRNA between 0.012-3.86 nM.
  • the siRNA provided in the present disclosure can inhibit FXI mRNA expression in HepG2 cells up to 86.9% FXI mRNA inhibition rate at a concentration of 50 nM.
  • the siRNA, pharmaceutical composition or siRNA conjugate provided in the present disclosure may have higher stability and/or higher activity in vivo. In some embodiments, the siRNA, pharmaceutical 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. In some embodiments, the siRNA, pharmaceutical composition or siRNA conjugate provided in the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo % FXI gene expression inhibition rate.
  • the siRNA, pharmaceutical 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 FXI gene expression in the liver. In some embodiments, the siRNA, pharmaceutical 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 FXI gene expression in liver in animal models.
  • the siRNA, pharmaceutical 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 FXI gene expression in liver in% of human subjects.
  • the siRNA conjugate provided in the present disclosure can inhibit FXI mRNA expression in mice at a concentration of 5 mg/kg, and the inhibition rate can reach 95.0%.
  • the siRNA conjugate provided in the present disclosure inhibits the expression of human FXI mRNA in humanized mice at a siRNA concentration of 3 mg/kg, the inhibition rate can reach 93.09%, and it can significantly inhibit plasma FXI.
  • the protein concentration, the inhibition rate can reach about 99%.
  • the siRNA conjugate provided in the present disclosure can significantly prolong the detection value of plasma APTT in CD57 mice, for example, by 64.9%.
  • the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present disclosure does not show significant off-target effects.
  • Off-target effects may be, for example, suppression of normal expression of genes other than target genes. It is believed that if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% compared to the target gene effect, the off-target effect is not significant.
  • siRNA, pharmaceutical composition and siRNA conjugate provided in the present disclosure can inhibit the expression of FXI gene, effectively treat and/or prevent thrombotic diseases and/or ischemic stroke symptoms caused by overexpression of FXI gene, Has good application prospects.
  • FXI mRNA refers to the mRNA with the sequence shown in Genbank registration number NM_000128.3.
  • target gene used in the present disclosure refers to the gene that transcribes the aforementioned FXI mRNA
  • target mRNA refers to the aforementioned FXI mRNA.
  • the capital letters C, G, U, A represent the base composition of nucleotides;
  • the lowercase letter m represents that the adjacent nucleotide to the left of the letter m is a methoxy group Modified nucleotides;
  • a lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide;
  • a lowercase letter s indicates that between the two adjacent nucleotides to the left and right of the letter s It is a phosphorothioate group connection;
  • P1 indicates that the adjacent nucleotide to the right of P1 is a 5'-phosphate nucleotide or a 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 nucleotide modified by vinyl phosphate (5'-(E)-vinylphosphonate, E
  • the letter combination Ps represents the adjacent nucleoside on the right side of the letter combination Ps
  • the acid is a phosphorothioate modified nucleotide.
  • the capital letter P indicates that the adjacent nucleotide to the right of the letter P is a 5'-phosphate nucleotide.
  • fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is replaced by fluorine
  • non-fluoro-modified nucleotide refers to Nucleotides or nucleotide analogs formed by substituting a non-fluorine group for the hydroxyl group at the 2'position of the ribose group of a nucleotide.
  • Nucleotide analogue refers to a nucleic acid that can replace nucleotides, but has a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymus The group of pyrimidine deoxyribonucleotides. Such as heteronucleotide, bridged nucleotide (BNA) or acyclic nucleotide.
  • the "methoxy-modified nucleotide” refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the expressions "complementary” or “reverse complement” can be used interchangeably, and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are connected to the other strand. The bases on are paired in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) matches.
  • Each base pair includes a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, bases at corresponding positions are not paired in a complementary manner.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” “Means that there is no more than one base mismatch between two nucleotide sequences; “complete reverse complement” means that there is no base mismatch between two nucleotide sequences.
  • nucleotide difference between 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 The type and sequence of nucleotides in the prepared siRNA or siRNA conjugate, the modified or unmodified nucleoside phosphoramidite monomer used in the solid phase synthesis of phosphoramidites (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites).
  • Phosphoramidite solid phase synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in the present disclosure are all commercially available.
  • siRNA conjugate means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; correspondingly, “conjugate” is Refers to the compound formed by covalent linkage between the various chemical parts.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugate should be understood as the general term of siRNA conjugate, the general term of siRNA conjugate represented by formula (305) and formula (307), or formula (305), formula (307), formula (308) SiRNA conjugate shown.
  • conjuggated molecule should be understood as a specific compound that can be conjugated to siRNA through a reaction, and finally form the siRNA conjugate of the present disclosure.
  • alkyl refers to straight and branched chains having a specified number of carbon atoms, the number is usually 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 Or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl groups include 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 including n-butyl, sec-butyl , Isobutyl and tert-butyl; "propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to residues that are the same as alkyl but have two points of attachment.
  • alkenyl refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon double bond, which is obtained from adjacent carbon atoms of the parent alkyl group. Obtained by removing one molecule of hydrogen. The group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl Group), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-ene-1- But-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc.
  • alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon triple bond, 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, etc.
  • an alkynyl group has 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 carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is completely unsaturated, that is, contains a ring according to Hückel theory , Delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups.
  • 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 halogen 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, or 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 or sulfur. Unless otherwise specified in the specification, heterocyclic groups are monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, and may include fused or bridged ring systems. 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 protective group makes the chemical functional group insensitive to specific reaction conditions, and can be added to and removed from the functional group in the molecule without substantially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d, John Wiley & Sons, New York, 1991, for reference In this way, the above-mentioned documents are incorporated into this article as a whole.
  • the protecting group is stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) or 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) or 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, cattle, sheep, rats, or any kind 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.
  • therapeutic benefits are obtained by eradicating or improving one or more physical 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 specific 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 ninth siRNA capable of inhibiting FXI gene expression. They will be described in detail below in turn.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and those skilled in the art know that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.
  • the siRNA may be the first siRNA.
  • the first siRNA contains a sense strand and an antisense strand, each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I 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 U
  • Z 2 is A
  • 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 is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and/or the nucleotide sequence II and SEQ ID NO: No more than one nucleotide difference between the nucleotide sequences shown in 2.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes the difference at position Z 4 , and Z 4 is selected from U, C or G. In some embodiments, the nucleotide difference is a difference at the Z 4 position, and Z 4 is selected from U, C, or G. In some embodiments, Z 3 is a nucleotide that is complementary to Z 4 . SiRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibition ability, and these siRNAs containing nucleotide differences are also within the protection scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse 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 U, Z 4 is A;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the 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 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, and the second nucleotide sequence refers to the sequence ID of the target mRNA.
  • the nucleotide sequence represented by NO:1 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 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 acid sequence III is CU, and the base composition of nucleotide sequence IV is AG; 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 UCU, and the base composition of nucleotide sequence IV is AGA; 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 UUCU, the base composition of nucleotide sequence IV is AGAA; 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, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CU , The base composition of nucleotide sequence IV is AG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • the siRNA may be a second siRNA.
  • the second siRNA contains a sense strand and an antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 61 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 62 The length is equal, and no more than 3 nucleotide differences:
  • Z 5 is U
  • Z 6 is A
  • 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 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.
  • nucleotide sequence II and SEQ ID NO: nucleotide differences between the nucleotide sequence shown at 62 include differences in location Z 8, and Z 8 is selected from U, C, or G.
  • the nucleotide difference is a difference at the Z 8 position, and Z 8 is selected from U, C, or G.
  • Z 7 is a nucleotide that is complementary to Z 8 .
  • SiRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibition ability, and these siRNAs 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: 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, and Z 8 is a nucleotide complementary to Z 7 ; in some embodiments Where Z 7 is U, Z 8 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,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 61 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is 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 AG, and the base composition of nucleotide sequence IV is CU; 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 AAG
  • the base composition of nucleotide sequence IV is CUU
  • 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 GAAG
  • the base composition of nucleotide sequence IV is CUUC; 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
  • the base composition of the nucleotide sequence III is AG according to the direction from the 5'end to the 3'end.
  • the base composition of nucleotide sequence IV is CU; 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, each nucleotide in the third siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and 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 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 1 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 as a difference at a position of Z 12, 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 mRNA inhibition ability, and these siRNAs containing nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 123
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124:
  • Z 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 A, Z 12 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: 121 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the core The base of nucleotide sequence IV is A; at this time, the length ratio of the sense strand and antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is GU, and 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; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is AGU, and the base composition of nucleotide sequence IV is ACU; At this
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is 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 siRNA may be the fourth siRNA.
  • the fourth siRNA contains a sense strand and an antisense strand, and 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, 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 U
  • Z 14 is A
  • 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 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.
  • nucleotide sequence II and SEQ ID NO: nucleotide differences between nucleotide sequence 182 includes difference 16 at a position Z, and the Z 16 is selected from U, C, or G.
  • the nucleotide difference is a difference at the Z 16 position, and Z 16 is selected from U, C or G.
  • Z 15 is a nucleotide that is complementary to Z 16 .
  • 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 In, Z 15 is U, Z 16 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,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 181 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the core The base of nucleotide sequence IV is A; at this time, the length ratio of the sense strand and antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is UU, and the base composition of nucleotide sequence IV is AA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is CUU, and the base composition of nucleotide sequence IV is AAG;
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UU , The base composition of nucleotide sequence IV is AA; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • 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, each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I 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 the nucleotide sequence shown in SEQ ID NO: 242 The length is equal, and no more than 3 nucleotide differences:
  • Z 17 is U
  • Z 18 is A
  • 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.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 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 U, C or G. In some embodiments, the nucleotide difference is a difference at the Z 20 position, and Z 20 is selected from U, 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 mRNA inhibition ability, and these siRNAs 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: 243
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 244:
  • the 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 In, Z 19 is U, Z 20 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,
  • 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 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the core The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is AA, and the base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is AAA, and the base composition of nucleotide sequence IV is UU
  • 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 AA , The base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • the siRNA may be a sixth siRNA.
  • the sixth siRNA contains a sense strand and an antisense strand, 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, 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 G
  • Z 22 is C
  • 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.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 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 U, G or A. In some embodiments, the difference is a difference at nucleotide position 24 Z, Z 24 is selected from U, G or A. In some embodiments, Z 23 is a nucleotide that is complementary to Z 24 . SiRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibition ability, and these siRNAs 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 G and Z 24 is C;
  • 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 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the core The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is GA, and the base composition of nucleotide sequence IV is UC; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is CGA, and the base composition of nucleotide sequence IV is UCG
  • 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 GA , The base composition of nucleotide sequence IV is UC; 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, 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: 361 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: 362 The length is equal, and 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.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 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 4 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at position Z 28 , and Z 4 is selected from A, C, or G. In some embodiments, Z 27 is a nucleotide that is complementary to Z 28 . SiRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibition ability, and these siRNAs 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 , Z 27 is A, Z 28 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.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, and the second nucleotide sequence refers to the sequence ID of the target mRNA.
  • 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 G, The base of nucleotide sequence IV is C; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is CG, and the base composition of nucleotide sequence IV is CG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is GCG, and the base composition of nucleotide sequence IV is CGC; At this time
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CG , The base composition of nucleotide sequence IV is CG; 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 U
  • Z 30 is A
  • 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 sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence shown in the nucleotide sequence SEQ ID NO: 421, and/or the nucleotide sequence II and SEQ ID NO There is no more than 1 nucleotide difference between the nucleotide sequences shown in :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 U, C or G. In some embodiments, the nucleotide difference is a difference at the Z 32 position, and Z 32 is selected from U, C, or G. In some embodiments, Z 31 is a nucleotide complementary to Z 32 . SiRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibition ability, and these siRNAs 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 , Z 31 is U, Z 32 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.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, and the second nucleotide sequence refers to the sequence ID of the target mRNA.
  • the nucleotide sequence represented by NO: 421 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 core The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is GA, and the base composition of nucleotide sequence IV is UC; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is AGA, and the base composition of nucleotide sequence IV is UCU
  • 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 GA , The base composition of nucleotide sequence IV is UC; 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 ninth siRNA.
  • the ninth siRNA contains a sense strand and an antisense strand, each nucleotide in the ninth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I and the nucleotide sequence shown in SEQ ID NO: 481 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 482 The length is equal, and no more than 3 nucleotide differences:
  • Z 33 is A
  • Z 34 is U
  • the nucleotide sequence I contains the nucleotide Z 35 whose position corresponds to Z 33
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 34 Acid Z 36
  • the Z 36 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 is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 481, and/or the nucleotide sequence II and SEQ ID NO: No more than one nucleotide difference between the nucleotide sequences shown in 2.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 482 includes the difference at position Z 36 , and Z 36 is selected from A, C or G. In some embodiments, the nucleotide difference as a difference at a position of Z 36, Z 36 is selected from A, C or G. In some embodiments, Z 35 is a nucleotide that is complementary to Z 36 . SiRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibition ability, and these siRNAs 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: 483, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 484:
  • Z 36 is the first nucleotide at the 5'end of the antisense strand, Z 35 is selected from A, U, G or C, and Z 36 is a nucleotide complementary to Z 35 ; in some embodiments Where Z 35 is A, Z 36 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.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, and the second nucleotide sequence refers to the sequence ID of the target mRNA.
  • the nucleotide sequence represented by NO: 481 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 U, and the core The base of nucleotide sequence IV is A; at this time, the length ratio of the sense strand and antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is CU, and the base composition of nucleotide sequence IV is AG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from 5'end to 3'end, the base composition of nucleotide sequence III is ACU, and the base composition of nucleotide sequence IV is AGU; At this time,
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CU , The base composition of nucleotide sequence IV is AG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • nucleotide sequence V, nucleic acid sequence, nucleotide modification in siRNA, and modification sequence is applicable to any one of the above-mentioned first siRNA to ninth 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, eighth siRNA and ninth siRNA are described one by one.
  • the siRNA also contains a nucleotide sequence V
  • 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 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, thus, the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure may be 19/21, 21/23, or 23. /25.
  • Each nucleotide in the nucleotide sequence V can be any nucleotide.
  • the nucleotide sequence V is two consecutive thymine deoxyribonucleotides ( dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to increase the affinity of the siRNA antisense strand with the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23. At this time, the siRNA of the present disclosure has better target mRNA silencing activity.
  • the nucleotide at the corresponding position of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to a nucleotide sequence of the target mRNA at the 5'end.
  • the nucleotide sequence of the target mRNA is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, or substantially reverse to nucleotide sequence composed of nucleotide sequence II and 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 3 is selected from A, U, G or C
  • Z 4 is a nucleotide complementary to Z 3 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 66.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68:
  • Z 8 is the first nucleotide at the 5'end of the antisense strand
  • Z 7 is selected from A, U, G or C
  • Z 8 is a nucleotide complementary to Z 7 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 125
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 126.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 127
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 128:
  • Z 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 .
  • 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 15 is selected from A, U, G, or C
  • Z 16 is a nucleotide complementary to Z 15 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 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 19 is selected from A, U, G or C
  • Z 20 is a nucleotide complementary to Z 19 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 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 23 is selected from A, U, G or C
  • Z 24 is a nucleotide complementary to Z 23 .
  • 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:
  • the 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 .
  • 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 31 is selected from A, U, G or C
  • Z 32 is a nucleotide complementary to Z 31 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 485, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 486.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 487
  • the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 488:
  • Z 36 is the first nucleotide at the 5'end of the antisense strand
  • Z 35 is selected from A, U, G, or C
  • Z 36 is a nucleotide complementary to Z 35 .
  • the siRNA described in the present disclosure is siFXIa1, siFXIa2, siFXIb1, siFXIb2, siFXIc1, siFXIc2, siFXId1, siFXId2, siFXIe1, siFXIe2, siFXIf1, siFXIf2, siFXIg1, siFXIg2, and siFXIg1 listed in Table 1a-1i , SiFXIh2siFXIi1 or siFXIi2.
  • 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 siRNAs of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups will not cause the siRNA conjugates of the present disclosure to inhibit the function of FXI gene expression Significantly weakened or lost.
  • the siRNA of the present disclosure contains at least 1 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 having Nucleotides of modified bases.
  • the modified nucleotides will not cause the siRNA to significantly weaken or lose the function of inhibiting gene expression.
  • At least one nucleotide in the sense strand or the antisense strand of the siRNA provided in the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate ester with a modified group 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 ribosyl group.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
  • the inventors of the present disclosure surprisingly discovered 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 in the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5'end to the 3'end, in the antisense strand
  • the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the antisense strand
  • the nucleotides at the remaining positions are non-fluorinated modified nucleotides.
  • fluoromodified nucleotides refer to nucleotides in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refer to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of a nucleotide with a non-fluorine group.
  • each non-fluorinated modified nucleotide is independently selected from among nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorinated group.
  • nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorinated group is independently selected from among nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorinated group.
  • nucleotides formed by replacing the hydroxyl group at the 2'position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotides are 2'-methoxy (2'-OMe) modified nucleotides, as shown in formula (8).
  • the 2'-substituted alkoxy-modified nucleotide can be, for example, a 2'-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (9) Show.
  • the 2'-amino (2'-NH 2 ) modified nucleotide 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, or acyclic nucleotides.
  • BNA Bridged Nucleic Acid
  • the 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, positions 7, 8, and 9 of the nucleotide sequence I Or the nucleotides at positions 5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand Wherein, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the antisense The nucleotides at the remaining positions in the chain are methoxy modified nucleotides.
  • the siRNA of the present disclosure is an siRNA with the following modifications: in the direction from the 5'end to the 3'end, the siRNA is at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand
  • the nucleotides are fluoro-modified nucleotides
  • the nucleotides in the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides
  • the siRNA in the direction from the 5'end to the 3'end
  • the nucleotides at positions 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand are fluorinated modified nucleotides
  • the nucleotides at the remaining positions 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
  • the nucleotides at the remaining positions of the chain are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, and 14th nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of siRNA are methoxy-modified nucleotides;
  • the nucleotides at positions 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the sense strand of the siRNA
  • the nucleotides at the remaining positions are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, 14th, and 16th nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at the position 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 siFXIa1-M1, siFXIa1-M2, siFXIa1-M3, siFXIa2-M1, siFXIa2-M2, siFXIa2-M3, siFXIb1-M1, siFXIb1 listed in Table 1a-Table 1i -M2, siFXIb1-M3, siFXIb2-M1, siFXIb2-M2, siFXIb2-M3, siFXIc1-M1, siFXIc1-M2, siFXIc1-M3, siFXIc2-M1, siFXIc2-M2, siFXIc2-M3, siFXId1-M1, siFXId1-M2 , SiFXId1-M3, siFXId2-M1, siFXId2-M2, siFXId2-M3, siFXId1-M1, siFXId1-M2 , SiFXId1-M3,
  • the modified siRNA is not only low in cost, but also makes the ribonuclease in the blood difficult to cut the nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.
  • the above-mentioned modified siRNA has a higher activity of inhibiting target mRNA.
  • the phosphate 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 having a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in 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 the siRNA and maintain the high specificity and affinity of base pairing.
  • the phosphorothioate group linkage exists in at least one of the following positions: the first and second cores of either end of the sense strand or the antisense strand Between nucleotides; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above.
  • the phosphorothioate group linkages are present at all of the above positions except the 5'end of the sense chain.
  • the phosphorothioate group linkages are present at all of the above 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 in the present disclosure is siFXIa1-M1S, siFXIa1-M2S, siFXIa1-M3S, siFXIa2-M1S, siFXIa2-M2S, siFXIa2-M3S, siFXIb1-M1S, siFXIb1 listed in Table 1a-Table 1i -M2S, siFXIb1-M3S, siFXIb2-M1S, siFXIb2-M2S, siFXIb2-M3S, siFXIc1-M1S, siFXIc1-M2S, siFXIc1-M3S, siFXIc2-M1S, siFXIc2-M2S, siFXIc2-M3S, siFXId , SiFXId1-M3S, siFXId2-M1S, siFXId2-M2S, siFXIc2-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 nucleotides are the nucleotides represented by formula (2) containing 5'-phosphate modifications, and the 5'-phosphate analogue modified nucleotides are those containing vinyl phosphate modifications.
  • the nucleotides are as shown in formula (3), or are phosphorothioate modified nucleotides, as shown in formula (5).
  • the siRNA provided in the present disclosure is siFXIa1-M1P1, siFXIa1-M2P1, siFXIa1-M3P1, siFXIa2-M1P1, siFXIa2-M2P1, siFXIa2-M3P1, siFXIa1-M1SP1, siFXIa1 listed in Table 1a-Table 1i -M2SP1, siFXIa1-M3SP1, siFXIa2-M1SP1, siFXIa2-M2SP1, siFXIa2-M3SP1, siFXIb1-M1P1, siFXIb1-M2P1, siFXIb1-M3P1, siFXIb2-M1P1, siFXIb1-M2P1, siFXIb2-M1P1, siFXIb1-M2P1, siFXIb2-M3P1, siFXIb1-M2SP1 , SiFXIb1-M2SP1,
  • 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 high target mRNA 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, the method for preparing nucleoside monomers with corresponding modifications and the introduction of modified nucleotide groups The siRNA method is also well known to those skilled in the art.
  • the present disclosure provides a pharmaceutical composition containing the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a carrier 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 (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
  • the content of siRNA and pharmaceutically acceptable carrier there is no special requirement for the content of siRNA and pharmaceutically acceptable carrier, and may be the conventional content of each component.
  • the weight ratio of 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 millosmole 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 not limited to subcutaneous, intramuscular or intravenous injection administration, and can also be but not limited to spray to the lungs or spray to other organs and tissues (such as liver) through the lungs.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter may also be referred to as an organic amine), auxiliary lipids and/or PEGylation Lipid.
  • the organic amine, auxiliary lipid and pegylated lipid may be selected from the amine-containing transfection compound described in CN103380113A (incorporated in its entirety by reference) or its pharmaceutically acceptable One or more of the accepted salt or derivative, auxiliary lipid, and pegylated lipid.
  • the organic amine may be a compound represented by formula (201) described in CN103380113A or a pharmaceutically acceptable salt thereof:
  • Each X 101 or X 102 is independently O, S, NA or CA, where A is hydrogen or a C 1 -C 20 hydrocarbon chain;
  • Each R 101 , R 102 , R 103 , R 104 , R 105 , R 106 or R 107 is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain lipid Group group, cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, substituted or unsubstituted, branched or straight chain acyl group, substituted Or unsubstituted, branched or straight chain aryl, substituted or unsubstituted, branched or straight chain heteroaryl;
  • x is an integer of 1-10;
  • R 103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
  • 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).
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group.
  • the radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 can be any of the following formulas (204)-(213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows R 103 and the formula (201)
  • the possible connection points of the nitrogen atom in 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 analogues and/or cholesterol derivatives
  • the pegylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-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).
  • the particles of the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, 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 weight of the siRNA and all lipids is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1: 18.
  • the weight ratio of 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, except that the siRNA provided in the present disclosure can replace the existing siRNA; in some In the embodiment, it can be prepared as follows:
  • the organic amine, auxiliary lipid and PEGylated 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 resulting 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, 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, for 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, polydispersity index is not higher than 0.30, osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be pH 7.2-7.6, encapsulation rate is not less than 90%, particle size is 60-100nm, more The dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution can be performed before, after or at the same time as removing impurities.
  • 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 can be non-covalently or covalently conjugated to the conjugating group, for example can 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 bonds.
  • 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.
  • connection methods can refer to 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 can 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.
  • the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated by reference This article.
  • the pharmaceutically acceptable targeting group may be selected from one or more of the following targeting molecules or ligands formed by their derivatives: lipophilic molecules, such as cholesterol, bile acids, Vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, and mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoprotein, asialosugar residues, lipoproteins (such as high density Lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitter (such as adrenaline), growth factor, transferrin, etc.
  • lipophilic molecules such as cholesterol, bile acids, Vitamins (such as vitamin E
  • each of the ligands is independently selected from a ligand capable of binding to cell surface receptors.
  • at least one ligand is a ligand capable of binding to a receptor on the surface of hepatocytes.
  • at least one ligand is a ligand capable of binding to a mammalian cell surface receptor.
  • at least one ligand is a ligand capable of binding to human hepatocyte surface receptors.
  • at least one ligand is a ligand capable of binding to the liver surface asialoglycoprotein receptor (ASGPR).
  • ASGPR liver surface asialoglycoprotein receptor
  • the pharmaceutically acceptable targeting group may be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells.
  • each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).
  • the ligand is a sugar or a derivative of sugar.
  • At least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar.
  • each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives.
  • each or at least one ligand is selected from the group consisting of the following sugars: glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives Food, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.
  • each of the ligands may be independently selected from D-mannanose, L-mannanose, D-arabinose, D-xylofuranose, L-xylofuranose, D- Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannanose, ⁇ -D-mannanose, ⁇ -D-mannanose, ⁇ -D-mannanose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-frutofuranose, ⁇ -D-fructose pyranose, ⁇ -D-pyranose Galactopyrose, ⁇ -D-galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-galactofuranose, glucosamine, sialic acid, galactosamine,
  • the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent , Second price, three price, four price. It should be understood that the monovalent, bivalent, trivalent, and tetravalent mentioned herein refer to the siRNA molecule and the conjugation group containing galactose or N-acetylgalactosamine as the targeting group to form siRNA conjugates.
  • the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3 or 1:4.
  • the pharmaceutically acceptable targeting group is N-acetylgalactosamine.
  • the siRNA described in the present disclosure is conjugated with a N-acetylgalactosamine-containing conjugating group, the N-acetylgalactosamine molecule is trivalent or tetravalent.
  • the siRNA described in the present disclosure is conjugated with a N-acetylgalactosamine-containing conjugating group, the N-acetylgalactosamine molecule is trivalent.
  • the targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • linkers, targeting groups, and connection methods with siRNA please refer to the disclosure of WO2015006740A2, and the entire content is incorporated herein by reference.
  • a suitable linker may have a structure as shown in formula (301):
  • k is an integer of 1-3;
  • L A having the formula (302) contains an amide bond-like portion of the structure shown, each of the L A at its two ends respectively of said group and the targeting moieties via ether linkages L C phase connection:
  • L B having the formula (303) comprises a pyrrolidine N- acyl chain portion shown structure, the linear portion having a carbonyl group at one end thereof and connected with the L C moiety through an amide bond, at the other end It has an oxygen group and is connected to the siRNA through a phosphate bond:
  • L C based aminomethane two monovalent 2-4 aminomethane or tris (hydroxymethyl) aminomethane linking group, via an oxygen atom of the L C L A portion of each of the phases by ether linkages connection, and connected by an amide bond via a nitrogen atom and L B of the portion.
  • the structure of the siRNA conjugate formed by N-acetylgalactosamine molecule and siRNA molecule is shown in the following formula (304):
  • the double helix structure represents siRNA.
  • 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.
  • the double helix structure represents the siRNA, and the linker is connected to the 3'end of the sense strand of the siRNA.
  • a suitable linker may have a structure as shown in formula (306):
  • l is an integer from 0-3;
  • # Indicates the site on the linker that is connected to the siRNA via a phosphate bond.
  • the siRNA conjugate has a structure as shown in formula (307):
  • the double helix structure represents the siRNA, and the linker is connected to the 3'end of the sense strand of the siRNA.
  • WO2015006740A2 describes in detail the preparation methods of various conjugates.
  • the siRNA conjugate of the present disclosure is obtained by a method well known to those skilled in the art.
  • WO2014025805A1 describes the preparation method of the structure represented by formula (305), Rajeev et al. described the preparation method of the structure represented by formula (307) in ChemBioChem2015, 16, 903-908.
  • the siRNA conjugate has a structure as shown in formula (308):
  • n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
  • Each m1, m2 or m3 is independently an integer selected from 2-10;
  • Each R 10 , R 11 , R 12 , R 13 , R 14 or R 15 is independently H, or is selected from the group consisting of: C 1 -C 10 alkyl, C 1 -C 10 haloalkyl and C 1 -C 10 alkoxy;
  • R 3 is a group represented by the formula A59:
  • E 1 is OH, SH or BH 2
  • Nu is the siRNA of the disclosure
  • L 1 may be selected from the group consisting of the A1-A26 group or any combination thereof, wherein the structure and definition of A1-A26 are as follows:
  • R' is C 1 -C 10 alkyl
  • Ra is selected from the group consisting of groups of formula A27-A45 or any combination thereof:
  • Rb is C 1 -C 10 alkyl; Represents the point at which the group is covalently attached.
  • L 1 is defined as a linear alkylene group for convenience, it may not be a linear group or have a different name, such as an amine or alkenyl group due to the above substitutions and/or substitutions.
  • the length of L 1 is the number of atoms in the chain connecting the two connection points.
  • a ring (such as a heterocyclylene or heteroarylene group) obtained by replacing the carbon atom of the linear alkylene group is counted as one atom.
  • each M 1 represents a targeting group, and its definition and selectable range are the same as the above-mentioned targeting group.
  • each M 1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
  • n1 may be an integer of 1-3
  • n3 may be an integer of 0-4
  • M 1 targeting groups in the conjugate is at least 2.
  • n1 + n3 ⁇ 2 so that the number of M 1 targeting groups is at least 3, thereby This makes it easier for the M 1 targeting group to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the conjugate to enter the cell through endocytosis.
  • n1 is an integer of 1-2
  • n3 is an integer of 0-1
  • n1+n3 2-3.
  • each m1, m2, or m3 is independently selected from an integer of 2-10, the spatial position between the multiple M 1 targeting groups can be adapted to the M 1 targeting group and the liver surface.
  • each R 10 , R 11 , R 12 , R 13 , R 14 or R 15 is independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, As well as one of the C 1 -C 10 alkoxy groups, the properties of the siRNA conjugate of the present disclosure will not be changed, and the objectives of the present disclosure can be achieved.
  • each R 10 , R 11 , R 12 , R 13 , R 14 or R 15 is each independently selected from H, methyl, or ethyl.
  • each of R 10 , R 11 , R 12 , R 13 , R 14, and R 15 is H.
  • R 3 is a group represented by the formula A59, where E 1 is OH, SH or BH 2. Based on the availability of raw materials for preparation, in some embodiments, E 1 is OH or SH.
  • R 2 The choice of R 2 is to realize the connection with the N atom on the nitrogen-containing skeleton and A59.
  • nitrogen-containing skeleton refers to a chain structure in which carbon atoms to which R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are connected and N atoms are connected to each other. Therefore, R 2 may be any linking group capable of linking the A59 group to the N atom on the nitrogen-containing skeleton in an appropriate manner.
  • the siRNA conjugate represented by formula (308) is prepared by a solid-phase synthesis process, the R 2 group needs to also contain a connection site to the N atom on the nitrogen-containing backbone.
  • connection site to the P atom in R 3 the site connected to the N atom on the nitrogen-containing backbone in R 2 forms an amide bond with the N atom, and the site connected to the P atom on R 3 forms a phosphate bond with the P atom ;
  • R 2 can be B5, B6, B5' or B6':
  • the value range of q 2 can be an integer of 1-10, and in some embodiments, q 2 is an integer of 1-5.
  • L 1 is to connect the M 1 targeting group to the N atom on the nitrogen-containing backbone to provide the liver targeting function for the siRNA conjugate represented by formula (308).
  • L 1 is selected from a linked combination of one or more of the groups of formula A1-A26.
  • L 1 is selected from a connection combination of one or more of A1, A4, A5, A6, A8, A10, A11, and A13.
  • L 1 is selected from a connected combination of at least two of A1, A4, A8, A10, and A11.
  • L 1 is selected from a connection combination of at least two of A1, A8, and A10.
  • the length of L 1 may be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms. In some embodiments, the length of L 1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atom.
  • j1 is an integer from 2-10, and in some embodiments, j1 is an integer from 3-5. In some embodiments, j2 is an integer from 2-10, and in some embodiments, j2 is an integer from 3-5.
  • R' is a C 1 -C 4 alkyl group, and in some embodiments, R'is one of methyl, ethyl, and isopropyl.
  • Ra is one of A27, A28, A29, A30, and A31. In some embodiments, Ra is A27 or A28.
  • Rb is a C 1 -C 5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl.
  • j1, j2, R', Ra, Rb are selected in each of formulas A1-A26, so as to realize that the M 1 targeting group is connected to the N atom on the nitrogen-containing backbone, and the M 1 target
  • the spatial position between the directional groups is more suitable for the M 1 targeting group to bind to the asialoglycoprotein receptor on the liver surface.
  • the conjugate has formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412 ), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):
  • the P atom in formula A59 can be attached to any possible position in the siRNA sequence, for example, the P atom in formula A59 can be attached to any nucleotide in the sense strand or antisense strand of the siRNA; In some embodiments, the P atom in formula A59 is attached to any nucleotide in the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is connected to the end of the siRNA sense strand or antisense strand; in some embodiments, the P atom in formula A59 is connected to the end of the siRNA sense strand. The end refers to the first 4 nucleotides from one end of the sense strand or the antisense strand.
  • the P atom in formula A59 is attached to the end of the siRNA sense strand or antisense strand; in some embodiments, the P atom in formula A59 is attached to the 3'end of the siRNA sense strand.
  • the siRNA conjugate represented by formula (308) enters the cell, when unwinding, a separate siRNA antisense strand can be released to block FXI mRNA translation The protein process inhibits FXI gene expression.
  • the P atom in formula A59 can be attached to any possible position on the nucleotide in the siRNA, for example, the 5'position of the nucleotide, the 2'position of the nucleotide, the 3 of the nucleotide. 'Position or nucleotide base.
  • the P atom in formula A59 can be connected to the 2'position, 3'position or 5'position of the nucleotide in the siRNA by forming a phosphodiester bond.
  • the P atom in formula A59 is connected to the oxygen atom formed after the 3'hydroxyl group of the 3'terminal nucleotide of the siRNA sense strand is dehydrogenated (at this time, the P atom in A59 can also be regarded as siRNA
  • the P atom in the phosphate group contained in A59), or the P atom in formula A59 is connected to the nucleotide by substituting the hydrogen in the 2'-hydroxyl group of one nucleotide in the sense strand of siRNA, or the P in formula A59
  • the atom is connected to the nucleotide by replacing the hydrogen in the 5'hydroxyl group of the 5'terminal nucleotide of the siRNA sense strand.
  • the inventors of the present disclosure unexpectedly discovered that the siRNA conjugates of the present disclosure have significantly improved plasma stability and low off-target effects, while also exhibiting higher FXI mRNA silencing activity.
  • the siRNA of the present disclosure may be one of the siRNAs shown in Tables 1a-1i. SiRNA conjugates containing these siRNAs show higher FXI mRNA silencing activity.
  • Table 1a The first siRNA sequence of the present disclosure
  • Table 1d The fourth siRNA sequence of the present disclosure
  • Table 1g The seventh siRNA sequence of the present disclosure
  • the capital letters C, G, U, A indicate the base composition of nucleotides;
  • 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 The adjacent nucleotide on the left side 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 are connected by phosphorothioate groups;
  • P1 indicates the right side of the P1
  • the adjacent nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • P1 represents a specifically modified VP, Ps, or P, where the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is a nucleotide modified by vinyl phosphate.
  • Ps indicates that the adjacent nucleotide to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the adjacent nucleotide to the right of the letter P is a 5'-phosphate nucleotide .
  • 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 NH 4 + , 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, and the siRNA or siRNA conjugate described in the present disclosure is sodium salt or partial sodium salt.
  • 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.
  • the siRNA conjugate represented by formula (308) can be prepared by the following method, which includes under the conditions of phosphoramidite solid phase synthesis, according to the nucleotides of the sense strand and antisense strand of the siRNA, respectively. Type and order, according to the direction of 3'to 5', connect the nucleoside monomers in turn.
  • the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization; isolate the sense strand of siRNA And the antisense strand, annealing, wherein the siRNA is the above-mentioned siRNA of the present disclosure;
  • the method also includes contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence linked to a solid support in the presence of coupling reaction conditions and a coupling reagent, so that the formula (321) The compound shown in) is linked to the nucleotide sequence through a coupling reaction.
  • the compound represented by formula (321) is also referred to as a conjugate molecule.
  • R 4 is a group capable of binding to the siRNA represented by Nu in the compound represented by formula (308). In some embodiments, R 4 is a group capable of covalently bonding to the siRNA represented by Nu. In some embodiments, R 4 is a group capable of being conjugated to any functional group of the siRNA represented by Nu through a phosphodiester bond through a reaction;
  • Each S 1 is independently a group formed by replacing all active hydroxyl groups in M 1 with YCOO- groups, wherein each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, and monofluoromethyl
  • Y is independently selected from methyl, trifluoromethyl, difluoromethyl, and monofluoromethyl
  • Y is methyl.
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , and M 1 are as described above.
  • R 4 is to realize the connection with the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for the synthesis of the siRNA conjugate represented by formula (308).
  • R 4 includes an R 2 linking group or a protected R 2 linking group, and a functional group that can react with siRNA to form the structure shown in A59.
  • R 4 includes a first functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu and a second functional group that can react with a hydroxyl or amino group to form a covalent bond, or contains The solid phase carrier connected by the covalent bond.
  • the first functional group is phosphoramidite, hydroxyl or protected hydroxyl.
  • the second functional group is phosphoramidite, carboxyl or carboxylate.
  • the second functional group is a solid support connected to other parts of the molecule via a covalent bond, and the covalent bond is formed by a hydroxyl group or an amino group.
  • the solid support is connected via a phosphate bond, a carboxylate bond, or an amide bond.
  • the solid phase carrier is a resin.
  • the first functional group contains a hydroxyl group, -OR k, or a group represented by formula (C3);
  • the second functional group contains formula (C1), (C2), (C3), (C1' ) Or (C3'):
  • q 1 is an integer from 1 to 4
  • X is O or NH
  • M + is a cation
  • R k is a hydroxyl protecting group
  • SPS is a solid phase carrier
  • the first functional group contains a phosphoramidite group, as shown in formula (C3)
  • the phosphoramidite group can be combined with a hydroxyl group at any position on the nucleotide, such as the 2'hydroxyl group or
  • the 3'hydroxyl group undergoes a coupling reaction to form a phosphite, which is oxidized or vulcanized to form a phosphodiester bond or a phosphorothioate bond represented by formula A59, and the conjugation molecule is conjugated to the siRNA.
  • the compound represented by formula (321) can be conjugated to nucleotides without affecting the acquisition of the siRNA conjugate represented by formula (308).
  • the compound represented by formula (321) is reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and In the subsequent oxidation or vulcanization process, phosphodiester linkage or phosphorothioate linkage is formed, and the compound represented by formula (321) is conjugated to siRNA.
  • the first functional group contains a protected hydroxyl group.
  • the second functional group includes a group that can react with a solid-phase support, and the reaction provides a conjugate molecule that includes the solid-phase support.
  • the second functional group contains a carboxyl group, carboxylate or phosphoramidite, as shown in formula (C1), (C2) or (C3), when the second functional group contains a carboxyl group or carboxylate.
  • the compound represented by the formula (321) and the solid phase carrier such as the hydroxyl or amino group on the resin, undergo an esterification reaction or amidation reaction to form a conjugated molecule containing the solid phase carrier connected via a carboxylate bond.
  • the compound represented by formula (321) undergoes a coupling reaction with a general solid phase carrier, such as a hydroxyl group on a resin, and is oxidized to form a solid phase containing a phosphodiester bond. Conjugation molecule of carrier. Subsequently, starting from the product after connecting the solid phase carrier, the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or the antisense strand of the siRNA with the conjugation group attached. In the process of phosphoramidite solid-phase synthesis, the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under coupling reaction conditions.
  • a general solid phase carrier such as a hydroxyl group on a resin
  • the first functional group contains a hydroxyl group or a protected hydroxyl group
  • the second functional group contains a solid-phase carrier connected via a carboxylate bond or a solid-phase carrier connected via an amide bond or a phosphate bond
  • the solid phase carrier as shown in formula (C1') or (C3').
  • the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA with the conjugation group attached. .
  • the carboxylate may be expressed as -COO - M + , where M + is a cation, for example, one selected from the group consisting of metal cation, ammonium cation NH 4 + , and organic ammonium cation.
  • M + is a cation, for example, one selected from the group consisting of metal cation, ammonium cation NH 4 + , and organic ammonium cation.
  • the metal ion is selected from one of alkali metal ions, such as K + or Na + .
  • the organic ammonium ion is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed by triethylamine or N,N-diamine Ammonium ion formed by isopropylethylamine.
  • the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.
  • R 4 contains the structure represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11') or (B12'):
  • q 1 is an integer of 1-4
  • q 2 is an integer of 1-10
  • X is O or NH
  • M + is a cation
  • R k is a hydroxyl protecting group
  • SPS is a solid phase carrier
  • q 1 is 1 or 2.
  • q 2 is an integer from 1-5.
  • R 4 contains a structure represented by formula (B9) or (B10).
  • R 4 contains a structure represented by formula (B11) or (B12).
  • R k is Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-bismethoxytrityl), TMTr (4 ,4',4"-trimethoxytrityl).
  • R k may be DMTr, that is, 4,4'-bismethoxytrityl ( 4,4'-dimethoxytrityl).
  • L 1 The definition of L 1 is as described above.
  • L 1 is used to connect the M 1 targeting group to the N atom on the nitrogen-containing backbone, thereby providing the liver targeting function for the siRNA conjugate represented by formula (308).
  • L 1 comprises any one of A1-A26 or a combination thereof.
  • the first functional group and the optional second functional group can be used to connect the conjugated molecule.
  • the siRNA conjugate represented by formula (308) to any possible position of the nucleotide sequence for example, the conjugate molecule is connected to the end of the nucleotide sequence, and the conjugate molecule is connected to the end of the nucleotide sequence.
  • the reaction conditions and reagents involved in the phosphoramidite nucleic acid solid-phase synthesis method known in the art are also applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.
  • each S 1 is independently M 1 . In some embodiments, each S 1 is independently a group formed by protecting at least one active hydroxyl group in M 1 by a hydroxyl protecting group. In some embodiments, each S 1 is independently a group formed by any active hydroxyl groups present in M 1 being protected by a hydroxyl protecting group. In some embodiments, any hydroxyl protecting group known to those skilled in the art can be used to protect the active hydroxyl group in M 1 .
  • the protected hydroxyl group can be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C 1 -C 10 alkyl and C 6 -C 10 aryl, and the C The 1- C 10 alkyl group and the C 6 -C 10 aryl group are optionally substituted with one or more substituents selected from the group consisting of halogen and C 1 -C6 alkyl.
  • each Y is independently selected from the group consisting of: methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl , Monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C 1 -C 6 alkyl phenyl.
  • each S 1 is independently selected from the group consisting of formula A46-A54:
  • S 1 is formula A49 or A50.
  • each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, normal One of propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.
  • the preparation method of the siRNA conjugate represented by formula (308) also includes the following steps: synthesizing another strand of siRNA (for example, when the above step synthesizes the siRNA sense strand to which the conjugate molecule is connected, also Including the synthesis of the antisense strand of siRNA according to the solid-phase synthesis method, and vice versa), separation of the sense strand and antisense strand, and annealing.
  • the solid-phase carrier connected to the nucleotide sequence and/or the conjugate molecule is cleaved, and at the same time the necessary protective group is removed (at this time, the compound represented by formula (321)
  • Each S 1 group is converted into the corresponding M 1 targeting group), and the siRNA sense strand (or antisense strand) and the corresponding antisense strand (or sense strand), sense strand and antisense strand connected with the conjugate molecule are obtained
  • the strands are annealed to form a double-stranded RNA structure, and the siRNA conjugate represented by formula (308) is obtained.
  • the preparation method of the siRNA conjugate represented by formula (308) comprises the following steps: in the presence of coupling reaction conditions and coupling reagents, the compound represented by formula (321) is combined with the sense strand or the reverse The first nucleoside monomer at the 3'end of the sense strand is contacted to connect the compound represented by formula (321) to the first nucleotide in the sequence.
  • nucleoside monomers are sequentially connected in the 3'to 5'direction to synthesize the sense strand or antisense strand of siRNA; wherein, the compound represented by formula (321) is R 4 A compound containing a first functional group and a second functional group, the first functional group contains a protected hydroxyl group, and the second functional group has the structure shown in formula (C1') or (C3'), before being connected to the first nucleoside monomer ,
  • the compound represented by formula (321) is deprotected; the connection of each nucleoside monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfurization; the sense strand or reverse of the nucleic acid connected with the conjugating group is obtained.
  • the nucleoside monomers are sequentially connected in the 3'to 5'direction to synthesize the antisense strand of the nucleic acid Or sense strand; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; deprotection group and cutting with solid phase carrier, separation and purification to obtain sense strand and antisense strand, annealing .
  • the preparation method of the siRNA conjugate represented by formula (308) includes the following steps: according to the nucleotide type and sequence of the sense strand or the antisense strand in the double-stranded siRNA, in accordance with 3'to 5'
  • the nucleoside monomers are connected in order to synthesize the sense strand and the antisense strand.
  • the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization to obtain a solid-phase carrier.
  • the sense strand and the antisense strand attached to the solid phase carrier in the presence of coupling reaction conditions and coupling reagents, the compound represented by formula (321) and the sense strand attached to the solid phase carrier or connected to the solid phase
  • the antisense strand on the carrier is contacted to connect the compound represented by formula (321) to the sense strand or the antisense strand, wherein the compound represented by formula (321) contains the first functional group in R 4 and the first functional group is phosphoramidite
  • the compound represented by formula (321) of the group remove the protective group and cut with the solid-phase carrier, separate and purify separately, obtain the sense strand or antisense strand of siRNA, and anneal, wherein the sense strand or antisense strand of the siRNA A conjugating group is attached to it.
  • the P atom in formula A59 is connected to the 3'end of the sense strand in the siRNA, and the preparation method of the siRNA conjugate represented by formula (308) includes:
  • the method for removing the protective group R k in the compound represented by the formula (321) includes contacting the compound represented by the formula (321) with a deprotection reagent under deprotection conditions.
  • the deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, the deprotection reagent may be selected from trifluoroacetic acid One or more of trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, in some embodiments, dichloroacetic acid.
  • the molar ratio of the deprotection reagent to the compound represented by formula (321) is 10:1 to 1000:1, and in some embodiments, 50:1 to 500:1.
  • the coupling reaction conditions and coupling reagents can use any conditions and reagents suitable for the aforementioned coupling reaction. In some embodiments, the same conditions and reagents as the coupling reaction in the solid phase synthesis method used can be used.
  • the conditions of the coupling reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C.
  • the molar ratio of the compound represented by formula (321) to the nucleoside monomer is 1:1-1:50, in some embodiments 1:2-1:5; the compound represented by formula (321) and the coupling reagent
  • the molar ratio can be 1:1-1:50, in some embodiments 1:3-1:10, and the reaction time is 200-3000 seconds, and in some embodiments 500-1500 seconds.
  • the coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, in some embodiments 5-ethylsulfide Base 1H-tetrazolium.
  • the coupling reaction can be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments is anhydrous acetonitrile.
  • the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
  • step (2) by the method of phosphoramidite nucleic acid solid-phase synthesis, starting from the nucleoside monomer prepared by the above steps and connected to the solid-phase carrier by the conjugation molecule, the third is synthesized according to the 3'-5' direction.
  • the conjugating group is attached to the 3'end of the resulting sense chain.
  • conditions for the solid-phase synthesis described in steps (2) and (3) include deprotection conditions of nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, and capping reaction conditions.
  • deprotection conditions of nucleoside monomers include deprotection conditions of nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, and capping reaction conditions.
  • the conditions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidizing reagents, vulcanization reaction conditions, and types and amounts of vulcanization reagents adopt various reagents, amounts and conditions conventionally used in the art.
  • the solid-phase synthesis in steps (2) and (3) may use the following conditions:
  • the nucleoside monomer deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, the deprotection reagent can be selected From one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, dichloroacetic acid.
  • the molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support can be 2:1-100:1, and in some embodiments, 3:1-50:1 .
  • the coupling reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, and the molar ratio of the nucleic acid sequence linked to the solid support to the nucleoside monomer can be 1:1-1:50. In some embodiments, it is 1:5-1:15; the molar ratio of the nucleic acid sequence connected to the solid-phase carrier and the coupling reagent is 1:1-1:100, and in some embodiments, it is 1:50-1:80
  • the reaction time and the choice of coupling reagents are the same as above.
  • the capping reaction conditions include a temperature of 0-50°C, 15-35°C in some embodiments, a reaction time of 5-500 seconds, and 10-100 seconds in some embodiments, and the selection of capping reagents is the same as described above.
  • the molar ratio of the total amount of capping reagent to the nucleic acid sequence linked to the solid phase carrier is 1:100-100:1, and in some embodiments, 1:10-10:1.
  • the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole
  • the molar ratio of acetic anhydride, N-methylimidazole and the nucleic acid sequence linked to the solid-phase carrier can be 1:1:10-10: 10:1, in some embodiments 1:1:2-2:2:1.
  • the oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and the oxidizing agent is iodine in some embodiments (In some embodiments, it is provided in the form of iodine water).
  • the molar ratio of the oxidizing reagent to the nucleic acid sequence linked to the solid support in the coupling step may be 1:1-100:1, and in some embodiments, 5:1-50:1.
  • the vulcanization reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, and in some embodiments 100-1000 seconds, the vulcanization reagent is hydrogenation in some embodiments. Xanthan.
  • the molar ratio of the vulcanizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is 10:1 to 1000:1, and in some embodiments, 10:1 to 500:1.
  • the method also includes separating the sense strand and antisense strand of the siRNA.
  • the separation method is well known to those skilled in the art, and generally includes cutting the synthesized nucleotide sequence from the solid support, removing the protective groups on the base, phosphate and ligand, purification and desalting .
  • Cleaving the synthesized nucleotide sequence from the solid support and removing the protective groups on the base, phosphate and ligand can be carried out according to the conventional cutting and deprotection methods in siRNA synthesis.
  • the obtained nucleotide sequence connected with the solid phase carrier is contacted with concentrated ammonia; in the process of deprotection, the protecting group YCOO- of the A46-A54 group is converted into a hydroxyl group, and the S 1 group is converted into the corresponding
  • the M 1 group produces the siRNA conjugate represented by formula (308).
  • the concentrated ammonia water can be 25-30% by weight ammonia water, and the amount of concentrated ammonia water can be 0.2ml/ ⁇ mol-0.8ml/ ⁇ mol compared with the target siRNA sequence.
  • the method further includes contacting the nucleotide sequence removed from the solid phase carrier with triethylamine trihydrofluoride to remove the 2'-TBDMS protection.
  • the corresponding nucleotide in the obtained target siRNA sequence has a free 2'-hydroxyl group.
  • the dosage of pure triethylamine trihydrofluoride can be 0.4ml/ ⁇ mol-1.0ml/ ⁇ mol. In this way, the siRNA conjugate represented by formula (308) can be obtained.
  • a preparative ion chromatography purification column can be used to complete the purification of nucleic acid through gradient elution of NaBr or NaCl; after the product is collected and combined, a reverse phase chromatography purification column can be used for desalting.
  • the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond between the nucleotides is basically bonded to the sodium ion, and the formula ( The siRNA conjugate shown in 308) basically exists in the form of sodium salt.
  • a well-known ion exchange method can be used to replace the sodium ions with hydrogen ions and/or other cations to obtain other forms of siRNA conjugates represented by formula (308). The cation is as described above.
  • the purity and molecular weight of the nucleic acid sequence can be tested at any time to better control the synthesis quality.
  • detection methods are well known to those skilled in the art.
  • the purity of nucleic acid can be detected by ion exchange chromatography, and the molecular weight can be determined by liquid mass spectrometry (LC-MS).
  • the annealing method is also well known to those skilled in the art.
  • the synthesized sense strand (S chain) and antisense strand (AS chain) can be simply mixed in an equimolar ratio, heated to 70-95°C in water for injection, and then cooled at room temperature to form a double bond through hydrogen bonding. Chain structure.
  • the siRNA conjugate represented by formula (308) can be obtained.
  • methods such as liquid-mass spectrometry can also be used to characterize the synthesized siRNA conjugate represented by formula (308) by means of molecular weight detection. It is determined that the synthesized siRNA conjugate is the siRNA conjugate represented by formula (308) of the target design, and the sequence of the synthesized siRNA is the sequence of the desired siRNA, for example, one of the sequences listed in Table 1.
  • the compound represented by formula (321) can be obtained by the following preparation method: the method includes combining the compound represented by formula (313) with a cyclic compound in an organic solvent, under esterification reaction conditions, and in the presence of a base and an esterification catalyst. Contact with acid anhydride, ion exchange, and separation to obtain the compound represented by formula (321):
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1, and S 1 are defined and selectable ranges as described above;
  • R 6 is a group that provides R 4 in formula (321); in some embodiments, R 6 has the structure shown in formula (A61):
  • R i is any group that can be connected to the N atom on the nitrogen-containing skeleton, connected to R k O and connected to a free hydroxyl group, and R k is a hydroxyl protecting group.
  • R 4 contains the first functional group and the second functional group as the hydroxyl protecting group, and the second functional group contains the structure represented by formula (C1) or (C2) as shown in formula (321) Compound.
  • the esterification reaction conditions include a reaction temperature of 0-100°C and a reaction time of 8-48 hours. In some embodiments, the esterification reaction conditions are a reaction temperature of 10-40°C and a reaction time of 20-30. hour.
  • the organic solvent includes epoxy-based solvents, ether-based solvents, halogenated alkane-based solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine One or more of.
  • the epoxy solvent is dioxane and/or tetrahydrofuran
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether
  • the halogenated alkane solvent is dichloromethane, trihydrofuran One or more of methyl chloride and 1,2-dichloroethane.
  • the organic solvent is dichloromethane. Relative to the compound represented by formula (313), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
  • the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments is succinic anhydride.
  • the molar ratio of the cyclic acid anhydride to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.
  • the esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine.
  • the molar ratio of the catalyst to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.
  • the base can be any inorganic base, organic base, or a combination thereof.
  • the base may be, for example, a tertiary amine.
  • the tertiary amine is triethylamine or N,N-diisopropylethylamine.
  • the molar ratio of the tertiary amine to the compound represented by formula (313) is 1:1-20:1, and in some embodiments, 3:1-10:1.
  • the ion exchange effect is to convert the compound represented by formula (321) into the desired form of carboxylic acid or carboxylate.
  • the method of ion exchange is well known to those skilled in the art, and suitable ion exchange solutions and exchange conditions can be used.
  • a conjugated molecule with M + cation is obtained, which is not described in detail here.
  • the ion exchange reaction is performed using a triethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is 0.2-0.8M.
  • the triethylamine phosphate solution The concentration of the triethylamine phosphate solution is 0.4-0.6M, relative to the compound represented by formula (313), the amount of the triethylamine phosphate solution is 3-6L/mol, in a further embodiment 4-5L/mol.
  • any suitable separation method can be used to separate the compound represented by formula (321) from the reaction mixture.
  • the solvent can be removed by evaporation, and then the compound represented by formula (321) can be separated by chromatography.
  • the solvent can be directly removed to obtain a crude product of the compound represented by formula (321), which can be directly used in subsequent reactions.
  • the preparation method of the compound represented by formula (321) further includes under condensation reaction conditions, in an organic solvent, in the presence of a condensation agent, a condensation catalyst and a tertiary amine, the above ion exchange reaction is obtained
  • the product is further contacted with a solid support containing amino or hydroxyl.
  • R 4 contains a first functional group and a second functional group
  • the first functional group contains a hydroxyl protecting group
  • the second functional group contains a compound represented by formula (321) having a structure represented by formula (C1′).
  • the solid-phase carrier is one of the carriers used in solid-phase synthesis of siRNA, some of which are well known to those skilled in the art.
  • the solid phase carrier may be selected from solid phase carriers containing active hydroxyl groups or amino functional groups.
  • the solid phase carrier is an amino resin or a hydroxyl resin.
  • the amino or hydroxyl resin has the following parameters: a particle size of 100-400 mesh (mesh), and a surface amino or hydroxyl loading of 0.2-0.5 mmol/g.
  • the dosage ratio of the compound represented by the formula (321) to the solid phase carrier is 10-400 ⁇ mol compound per gram of solid phase carrier ( ⁇ mol/g). In some embodiments, the dosage ratio of the compound represented by formula (321) to the solid phase carrier is 50-200 ⁇ mol/g.
  • the organic solvent may be any suitable solvent or mixed solvent known to those skilled in the art.
  • the organic solvent is acetonitrile, epoxy-based solvents, ether-based solvents, halogenated alkanes solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine.
  • the epoxy solvent is dioxane and/or tetrahydrofuran
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether
  • the halogenated alkane solvent is dichloromethane, trihydrofuran One or more of methyl chloride and 1,2-dichloroethane.
  • the organic solvent is acetonitrile. Relative to the compound represented by formula (321), the amount of the organic solvent is 20-200 L/mol, in some embodiments 50-100 L/mol.
  • the condensing agent may be benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate (PyBop), 3- Diethoxyphosphoryl-1,2,3-benzotriazin-4(3H)-one (3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one, DEPBT) and/or O- O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate), in some embodiments, the condensing agent is O-benzotriazol-tetramethyl Urea hexafluorophosphate.
  • the molar ratio of the condensing agent to the compound represented by formula (321) is 1:1-20:1, and in other embodiments, 1:1-5:1.
  • the tertiary amine is triethylamine and/or N,N-diisopropylethylamine, in some embodiments N,N-diisopropylethylamine; the tertiary The molar ratio of the amine to the compound represented by formula (321) is 1:1-20:1, and in some embodiments, 1:1-5:1.
  • the method for preparing the compound represented by formula (321) may also include contacting the obtained condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions to obtain formula (321).
  • the function of the capping reaction is to remove any reactive functional groups that have not yet been completely reacted, so as to avoid unnecessary by-products in subsequent reactions.
  • the conditions of the capping reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C, and a reaction time of 1-10h, and in some embodiments 3-6h.
  • the capping reagent can be the capping reagent used in siRNA solid-phase synthesis, and the capping reagent used in siRNA solid-phase synthesis is well known to those skilled in the art.
  • the capping reagent consists of capping reagent 1 (cap1) and capping reagent 2 (cap2), wherein capping reagent 1 is N-methylimidazole, and in some embodiments, it is Pyridine/acetonitrile is provided as a mixed solution, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1, in some embodiments 1:3-1:1, and the total volume of pyridine and acetonitrile is compared with N-methyl The volume ratio of imidazole is 1:1-10:1, and in some embodiments, 3:1-7:1.
  • the capping reagent 2 is acetic anhydride.
  • the capping reagent 2 is provided in the form of a solution of acetic anhydride in acetonitrile, wherein the volume ratio of acetic anhydride and acetonitrile is 1:1-1:10, and in further embodiments is 1:2-1 :6.
  • the ratio of the volume of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass of the compound represented by formula (321) is 5ml/g-50ml/g, in some embodiments 15ml/g g-30ml/g.
  • the ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound represented by formula (321) is 0.5ml/g-10ml/g, and in some embodiments is 1ml/g-5ml/g.
  • the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole.
  • the organic solvent is acetonitrile, epoxy-based solvents, ether-based solvents, halogenated alkanes solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine.
  • the organic solvent is acetonitrile. Relative to the compound represented by formula (321), the amount of the organic solvent is 10-50 L/mol, and in some embodiments, 5-30 L/mol.
  • the acylation catalyst may be selected from any catalyst that can be used for esterification condensation or amidation condensation, such as basic heterocyclic compounds.
  • the acylation catalyst is 4-dimethylaminopyridine.
  • the mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1 to 1:1, in some embodiments 0.01:1 to 0.1:1.
  • any suitable separation method may be used to separate the compound represented by formula (321) from the reaction mixture.
  • the compound represented by formula (321) can be obtained by fully washing with an organic solvent and filtering to remove unreacted reactants, excess capping reagents and other impurities.
  • the organic solvent is selected from acetonitrile, two Methyl chloride, methanol, in some embodiments acetonitrile.
  • the preparation method of the conjugate molecule represented by formula (321) includes combining the compound represented by formula (313) with phosphorous in an organic solvent, under coupling reaction conditions, and in the presence of a coupling reagent.
  • the diamide is contacted and separated to obtain the compound represented by formula (321).
  • R 4 contains a first functional group and a second functional group
  • the first functional group contains a hydroxyl protecting group
  • the second functional group contains a compound represented by formula (321) having a structure represented by formula (C3).
  • the coupling reaction conditions include that the temperature can be 0-50°C, for example 15-35°C, and the molar ratio of the compound represented by formula (313) to the phosphoramidite can be 1:1-1: 50, for example, 1:5-1:15; the molar ratio of the compound represented by formula (313) and the coupling reagent can be 1:1-1:100, for example, 1:50-1:80; the reaction time can be 200-3000 seconds, for example, 500-1500 seconds.
  • the phosphoramidite may be, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, which is commercially available or synthesized according to a method known in the art.
  • the coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, for example 5-ethylthio 1H-tetrazole Azole.
  • the coupling reaction can be carried out in an organic solvent, and the organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous methylene chloride, for example, anhydrous acetonitrile.
  • the amount of the organic solvent is 3-50 L/mol, for example, it may be 5-20 L/mol.
  • the hydroxyl group in the compound represented by the formula (313) reacts with the phosphoramidite to form a phosphoramidite group.
  • the solvent can be directly removed to obtain a crude product of the compound represented by formula (321), which can be directly used in subsequent reactions.
  • the preparation method of the compound represented by formula (321) further includes the following steps: under coupling reaction conditions, in an organic solvent, and in the presence of a coupling reagent, the separated product is further mixed with The solid support of the hydroxyl group is brought into contact. Subsequently, through capping reaction and oxidation reaction, the compound represented by formula (321) is isolated. In this case, obtained is R 4 contains a first functional group and a second functional group, the first functional group containing a hydroxyl protective group, the second functional group having the formula (321) the compound represented by the structure represented by the formula (C3 ').
  • the solid phase carrier is a solid phase carrier known in the art that can be used for nucleic acid solid phase synthesis, for example, it may be a commercially available general solid phase carrier after deprotection reaction ( HL UnyLinker TM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, structure shown in formula B80):
  • the deprotection reaction is well known to those skilled in the art.
  • the deprotection conditions include a temperature of 0-50°C, such as 15-35°C, and a reaction time of 30-300 seconds, such as 50-150 seconds.
  • the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid.
  • the deprotection reagent is dichloroacetic acid.
  • the molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the stationary phase is 2:1-100:1, for example, 3:1-50:1.
  • the coupling reaction conditions and the selection of coupling reagents can be as described above.
  • the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.
  • the capping reaction conditions include a temperature of 0-50°C, such as 15-35°C, a reaction time of 5-500 seconds, such as 10-100 seconds, and the capping reaction is performed in the presence of a capping reagent.
  • the selection and amount of capping reagent can be as described above.
  • the oxidation reaction conditions include a temperature of 0-50°C, such as 15-35°C, a reaction time of 1-100 seconds, such as 5-50 seconds, and an oxidizing agent such as iodine (in some embodiments, iodine Provided in the form of water).
  • an oxidizing agent such as iodine (in some embodiments, iodine Provided in the form of water).
  • the molar ratio of the oxidizing agent to the nucleic acid sequence linked to the solid phase carrier is 1:1-100:1, for example, it may be 5:1-50:1.
  • R 6 is one of the groups of formula B7 or B8,
  • the compound represented by formula (313) can be obtained by the following preparation method: in an organic solvent, under amidation reaction conditions, and in the presence of a condensing agent and tertiary amine for amidation reaction, the formula (314) The compound is contacted with the compound represented by formula (A-1) or the compound represented by formula (A-2), and then separated:
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , S 1 , q 2 and R k are each defined and selectable ranges as As mentioned earlier.
  • the amidation reaction conditions may include a reaction temperature of 0-100°C and a reaction time of 1-48 hours. In some embodiments, the amidation reaction conditions may include a reaction temperature of 10-40°C and a reaction time of 2 16 hours.
  • the organic solvent is an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diiso One or more of propylethylamine.
  • the alcohol solvent is one or more of methanol, ethanol, and propanol in some embodiments, and ethanol in some embodiments.
  • the epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments.
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments.
  • the halogenated alkane solvent is one or more of dichloromethane, chloroform, and 1,2-dichloroethane in some embodiments.
  • the organic solvent is dichloromethane. Relative to the compound represented by formula (314), the amount of organic solvent is 3-50 L/mol, and in a further embodiment, 3-20 L/mol.
  • the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2, 3-Benzazole 4(3H)-one, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxy Carbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazole-tetramethylurea hexafluorophosphate, in a further embodiment 3-diethoxyphosphoryl- 1,2,3-Benzazole 4(3H)-one.
  • the molar ratio of the amidation reaction condensing agent to the compound represented by formula (314) may be 1:1-10:1, and in some embodiments, 2.5:1-5:1.
  • the tertiary amine is triethylamine or N,N-diisopropylethylamine, and in further embodiments is N,N-diisopropylethylamine.
  • the molar ratio of the tertiary amine to the compound represented by formula (314) is 3:1-20:1, and in some embodiments, 5:1-10:1.
  • the compounds represented by formula (A-1) and formula (A-2) can be prepared in any suitable manner.
  • R k is a DMTr group
  • the compound represented by formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1,2-propanediol can be first contacted with cyclic anhydride , And then react with DMTrCl to prepare the compound represented by formula (A-2).
  • the cyclic anhydride may be a cyclic anhydride with 4-13 carbon atoms, and in some embodiments, 4-8.
  • the compound represented by formula (313) can also be prepared by sequentially reacting the compound represented by formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol and DMTrCl. It is easily understood by those skilled in the art that these modifications will not affect the structure and function of the compound represented by formula (313), and these modifications are easily implemented by those skilled in the art on the basis of the above methods.
  • any suitable separation method can be used to separate the compound represented by formula (313) from the reaction mixture.
  • the solvent can be removed by evaporation, and then the compound represented by formula (313) can be separated by chromatographic methods.
  • the solvent can be directly removed to obtain a crude product of the compound represented by formula (313), which can be directly used in subsequent reactions.
  • the compound represented by formula (314) can be obtained by the following preparation method: the method comprises in an organic solvent, in the presence of a condensing agent for amidation reaction and a tertiary amine, under condensation reaction conditions, the formula ( The compound represented by 320) is contacted with the compound represented by formula (316), and then separated:
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 have the same definitions and selectable ranges as described above.
  • the compound represented by formula (316) can be prepared by, for example, the compound disclosed in J. Am. Chem. Soc. 2014, 136, 16958-16961, or the compound represented by formula (316) can be prepared by a person skilled in the art by various methods
  • the method disclosed in Example 1 of U.S. Patent No. 8,106,022 B2 can be used to prepare certain compounds of formula (316), and the entire contents of the above documents are incorporated herein by reference.
  • the condensation reaction conditions include a reaction temperature of 0-100°C, a reaction time of 0.1-24 hours, and in some embodiments, a reaction temperature of 10-40°C, and a reaction time of 0.5-16 hours.
  • the organic solvent is acetonitrile, epoxy-based solvents, ether-based solvents, halogenated alkanes solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl
  • the epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments
  • the ether solvent is diethyl ether and/or methyl tert-butyl in some embodiments
  • the halogenated alkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane.
  • the organic solvent is two Methyl chloride. Relative to the compound represented by formula (320), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
  • the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2, 3-Benzazole 4(3H)-one (DEPBT), O-benzotriazole-tetramethylurea hexafluorophosphate/ester, 4-(4,6-dimethoxytriazin-2-yl) )
  • One or more of 4-methylmorpholine hydrochloride or 1-hydroxybenzotriazole in a further embodiment benzotriazol-1-yl-oxytripyrrolidinyl phosphonium A mixture of hexafluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazol-1-yl-oxytripyrrolidinyl phosphonium hexafluorophosphate/salt and 1-hydroxybenzotriazole are etc. Molar amount. The molar ratio of the total amidation reaction condensing agent to
  • the tertiary amine can be N-methylmorpholine, triethylamine or N,N-diisopropylethylamine, in some embodiments it is N-methylmorpholine; the tertiary amine is of the formula ( The molar ratio of the compound shown in 316) may be 2:1-10:1, and in some embodiments, 2:1-5:1.
  • any suitable separation method can be used to separate the compound represented by formula (314) from the reaction mixture.
  • the solvent can be removed by evaporation, and then the compound represented by formula (314) can be separated by chromatographic methods.
  • the solvent can be directly removed to obtain a crude product of the compound represented by formula (314), which can be directly used in subsequent reactions.
  • siRNA conjugate of the present disclosure can also be used in combination with other pharmaceutically acceptable excipients, which can be one or more of various formulations or compounds conventionally used in the art.
  • pharmaceutically acceptable excipients can be one or more of various formulations or compounds conventionally used in the art.
  • pharmaceutical composition please refer to the above about the present disclosure. Description of the pharmaceutical composition.
  • siRNA composition containing the siRNA and conjugate of the present disclosure
  • the present disclosure provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for the treatment and/or prevention of thrombotic diseases and/or ischemic stroke use.
  • the present disclosure provides a method for preventing and/or treating thrombotic diseases and/or ischemic stroke, the method comprising adding an effective amount of the siRNA and/or pharmaceutical composition of the present disclosure and/or The siRNA conjugate is administered to a subject in need.
  • the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure can be used to prevent and/or treat thrombotic diseases and/or ischemic stroke, or be used to prepare for the prevention and/or treatment of thrombotic Drugs for disease and/or ischemic stroke.
  • 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 methods of the present disclosure include local administration and systemic administration. Generally speaking, local administration results in the delivery of more siRNA conjugates to a specific site compared to the subject's systemic circulation; while systemic administration results in the delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure Basic systemic circulation to the subject.
  • an administration method capable of delivering drugs to the liver 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 buccal administration and sublingual administration).
  • the frequency of administration can be once or more daily, weekly, every two weeks, every three weeks, every month 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. May be in cell cultures or experimental animals Determination Toxicity and therapeutic efficacy by standard pharmaceutical procedures, e.g. assays and ED 50 dose (the amount of the reaction finger to cause 50% of the maximum intensity of the reaction of the LD 50 (so that dose lethal to 50% of the population died), In the qualitative response, it refers to the dose that can cause a positive response 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.
  • the amount of siRNA When administering the siRNA, pharmaceutical composition and/or siRNA conjugate described in the present disclosure, for example, for male or female C57BL/6N mice 6-12 weeks old and weighing 18-25 g, the amount of siRNA :
  • the amount of siRNA may 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, and others In one embodiment, it is 0.1-15 mg/kg body weight, and in other embodiments, it is 0.1-10 mg/kg body weight;
  • the amount of siRNA can be 0.001-50 mg /kg body weight, in some embodiments 0.01-10 mg/kg body weight, in some embodiments 0.05-5 mg/kg body weight, and in some embodiments 0.1-3 mg/kg body weight.
  • the present disclosure provides a method for inhibiting FXI gene expression in liver cells, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver.
  • Cell contact, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are introduced into the hepatocytes, and the purpose of inhibiting the FXI gene expression in the hepatocytes is achieved through the mechanism of RNA interference.
  • the liver cells may be selected from liver cancer cell lines such as SMMC-7721, HepG2, Huh7, or isolated primary liver cells.
  • the cell is a HepG2 liver cancer cell.
  • 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 expression of the target gene, and This results in an extracellular concentration of 1 pM to 1 ⁇ M, 0.01 nM to 100 nM, 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 include 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 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.
  • Lipofectamine TM 2000 (Invitrogen) is used as the transfection reagent, and the specific operation refers to the instructions provided by the manufacturer .
  • C57BL/6N mice 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., hereinafter referred to as C57 mice.
  • Heterozygous humanized mice 6-8 weeks old, purchased from Saiye Biotechnology Co., Ltd.
  • the conjugate L10-siFXIf1M1S was synthesized.
  • the conjugate is an siRNA conjugate formed by conjugating an L-9 conjugate molecule and siRNA numbered siFXIf1M1S. See Table 3 for the sequence of siRNA conjugated in this conjugate.
  • the L-10 compound was synthesized according to the following method:
  • GAL-1 N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Company, 463.8mmol
  • acetic anhydride purchased from Enox Company, 5565.6mmol
  • step (1-1-1a) Dissolve the GAL-2 (35.1g, 90.0mmol) obtained in step (1-1-1a) in 213ml of anhydrous 1,2-dichloroethane, add 24.0g TMSOTf in an ice water bath and under nitrogen protection (CAS number: 27607-77-8, purchased from Macleans Company, 108.0 mmol), react at room temperature overnight.
  • step (1-1-1b) The GAL-3 (26.9g, 81.7mmol) obtained in step (1-1-1b) was dissolved in 136ml of anhydrous 1,2-dichloroethane, and the dried 30g molecular sieve powder, then add 9.0g 5-hexen-1-ol (CAS number: 821-41-0, purchased from Adamas-beta company, 89.9mmol), stir at room temperature for 30 minutes, add under ice bath and nitrogen protection 9.08g TMSOTf (40.9mmol), stirred and reacted overnight at room temperature.
  • 9.0g 5-hexen-1-ol CAS number: 821-41-0, purchased from Adamas-beta company, 89.9mmol
  • the filtrate is diluted with 300ml dichloromethane, filtered with diatomaceous earth, and then 500ml saturated sodium bicarbonate aqueous solution is added and stirred for 10 minutes to wash, separate the organic phase, the aqueous phase is extracted once with 300ml dichloroethane, and the organic phases are combined They were washed with 300ml saturated sodium bicarbonate aqueous solution and 300ml saturated brine respectively, the organic phase was separated, dried with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 41.3g of yellow sugar thin product GAL-4, which was carried out directly without purification.
  • One-step oxidation reaction is carried out directly without purification.
  • GAL-4 (14.9g, 34.7mmol,) obtained according to the method described in step (1-1-1c) was dissolved in a mixed solvent of 77ml dichloromethane and 77ml acetonitrile, and 103ml deionized water and 29.7g were added respectively Sodium periodate (CAS number: 7790-28-5, purchased from Aladdin Company, 138.8mmol), stirred under ice water bath for 10 minutes, adding ruthenium trichloride (CAS number: 14898-67-0, purchased from An Nai Kyrgyzstan, 238mg, 1.145mmol), react at room temperature overnight.
  • the reaction solution was diluted with 300 ml of water and stirred, and saturated sodium bicarbonate was added to adjust the pH to about 7.5.
  • the organic phase was separated and discarded.
  • the aqueous phase was extracted with dichloromethane three times with 200 ml each time and the organic phase was discarded. Adjust the pH of the aqueous phase to about 3 with citric acid solid, extract three times with 200ml of dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 6.85g of white foamy solid product GAL-5 .
  • step (1-1-2) The L-8 (40g, 27.09mmol, obtained by combining multiple batches) obtained in step (1-1-2) and A-1 (41.418g, 81.27) obtained in step (1-1-3a) mmol), dissolve in 271ml of dichloromethane, add 3-diethoxyphosphoryl-1,2,3-benzoxazole 4(3H)-one (DEPBT) (24.318g, 81.37mmol), then add diiso Propylethylamine (21.007g, 162.54mmol), stirred for 1.5h at 25°C, washed the organic phase with 800ml saturated sodium bicarbonate, and extracted the aqueous phase with dichloromethane 3 times, each time with 50ml, washed with 150ml saturated brine The organic phase and the aqueous phase were extracted once with 50 ml of dichloromethane.
  • DEPBT 3-diethoxyphosphoryl-1,2,3-benzoxazole 4(3H)-one
  • the L-10 compound is prepared by attaching the L-9 conjugated molecule to the solid support.
  • HBTU O-benzotriazole-tetramethylurea hexafluorophosphate
  • DIEA diisopropylethylamine
  • the filter cake was rinsed with DCM twice, 300ml each time, and acetonitrile rinsed 3 times, each 300ml each time, vacuum oil pump drying for 18h, and then add the raw materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) according to the feeding ratio shown in Table 2 for capping reaction.
  • DMAP 4-dimethylaminopyridine
  • the L-10 compound (ie, the L-9 conjugated molecule connected to the solid support) was 102 g, and the loading was 90.8 ⁇ mol/g.
  • CapA and CapB are capping reagent solutions
  • CapA is a pyridine/acetonitrile mixed solution of 20% by volume N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5
  • CapB is a solution of 20% by volume of acetic anhydride in acetonitrile.
  • the L-10 compound prepared by the above steps starts the cycle, and connects the nucleoside monomers one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides.
  • Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Among them, when two nucleotides are connected by phosphate ester, when the next nucleoside monomer is connected, there are four steps of deprotection, coupling, capping, and oxidation. When two nucleotides are connected by phosphorothioate, when the next nucleoside monomer is connected, there are four steps of protection, coupling, capping and vulcanization.
  • the synthesis conditions are given as follows:
  • the nucleoside monomer is provided in 0.1M acetonitrile solution.
  • the conditions of the deprotection reaction in each step are the same, that is, the temperature is 25°C, the reaction time is 70 seconds, and the deprotection reagent is dichloroacetic acid in dichloromethane (3% v/v), the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.
  • each step of the coupling reaction is the same, including the temperature of 25°C, the molar ratio of the nucleic acid sequence connected to the solid-phase carrier to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid-phase carrier and the coupling reagent The ratio is 1:65, the reaction time is 600 seconds, and the coupling reagent is a 0.5 M acetonitrile solution of 5-(Ethylthio)-1H-tetrazole (ETT).
  • ETT Ethylthio-1H-tetrazole
  • the capping conditions for each step are the same, including a temperature of 25°C and a reaction time of 15 seconds.
  • the oxidation reaction conditions for each step are the same, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidizing reagent of 0.05M iodine water.
  • the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling step is 30:1.
  • each step of the vulcanization reaction is the same, including the temperature of 25°C, the reaction time of 300 seconds, and the vulcanizing reagent is hydroxanthin.
  • the molar ratio of the vulcanizing reagent to the nucleic acid sequence connected to the solid support in the coupling step is 120:1.
  • nucleic acid sequence connected on the solid-phase carrier is cut, deprotected, purified, and desalted in sequence, and then lyophilized to obtain the sense strand, where:
  • the cutting and deprotection conditions are as follows: add the synthesized nucleotide sequence linked to the carrier into 25wt% ammonia water, the amount of ammonia water is 0.5ml/ ⁇ mol, react at 55°C for 16h, filter to remove the remaining carrier, and remove the supernatant. Concentrate to dryness in vacuo.
  • eluent A 20mM sodium phosphate (pH 8.1)
  • elution gradient: eluent A: eluent B 100:0-50:50
  • the specific conditions include using a Sephadex G25 (Sephadex G25) as the filler and eluting with deionized water.
  • the detection method is as follows: ion exchange chromatography (IEX-HPLC) is used to detect the purity of the above-mentioned sense chain, and liquid mass spectrometry (LC-MS) is used to analyze the molecular weight.
  • IEX-HPLC ion exchange chromatography
  • LC-MS liquid mass spectrometry
  • the theoretical value is 7584.5 and the measured value is 7584.0.
  • the measured value is consistent with the theoretical value, indicating that the synthesized 3'end is the sense strand SS conjugated with an L-9 conjugate molecule.
  • the universal solid-phase carrier (UnyLinker TM loaded HL Solid Supports, Kinovate Life Sciences Company) initiated the cycle to synthesize the antisense strand of the conjugate L10-siFXIf1M1S.
  • the deprotection, coupling, capping, oxidation or vulcanization reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method are the same as the synthetic sense strand.
  • the antisense strand AS was obtained by freeze-drying.
  • the sense strand and the antisense strand were separately dissolved in water for injection to obtain a 40 mg/mL solution, mixed in an equimolar ratio, heated at 50°C for 15 minutes, and cooled at room temperature to obtain an annealed product. Lyophilized to obtain lyophilized powder.
  • Use ultrapure water (Milli-Q ultrapure water meter, resistivity 18.2M ⁇ *cm(25°C)) to dilute the conjugate to a concentration of 0.2mg/mL, then use liquid mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, Model: LCT Premier) for molecular weight detection.
  • the measured value is consistent with the theoretical value, indicating that the synthesized siRNA conjugate is a target designed double-stranded nucleic acid sequence with an L-9 conjugate molecule. Its structure is shown in formula (403).
  • the siRNA is the sequence shown in Table 3 corresponding to the conjugate L10-siFXIf1M1S.
  • the capital letters C, G, U, A indicate the base composition of nucleotides;
  • 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 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 of the letter s are connected by phosphorothioate groups;
  • the uppercase letter P indicates the letter The adjacent nucleotide to the right of P is a 5'-phosphate nucleotide.
  • siRNA conjugates of the present disclosure were further synthesized: L10-siFXIa1M1SP, L10-siFXIb1M1SP, L10-siFXIc1M1SP, L10-siFXId1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP, L10-siFXIg1M1SP, L10-siFXIg1M1L10 siFXIi1M1S and L10-siFXIi1M1SP.
  • sequences contained in these conjugates are siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIe1M1SP, siFXIg1M1SP, siFXIh1M1SP, siFXIi1M1S and siFXIi1M1SP in Table 3, respectively.
  • the difference between the preparation methods is only: (1) The siRNA sense strand and antisense strand sequence contained in each conjugate in Table 3 are used to replace the sense strand and antisense strand sequence of the conjugate L10-siFXIf1M1S; (2) For Conjugates L10-siFXIa1M1SP, L10-siFXIb1M1SP, L10-siFXIc1M1SP, L10-siFXId1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP, L10-siFXIh1M1SP and L10-siFXIh1M1S
  • the acid is a 5'-phosphonucleotide.
  • the process of preparing the antisense strand according to the solid-phase phosphoramidite method after connecting the last nucleoside monomer of the antisense strand, it is deprotected, coupled and capped. 4.
  • the four-step oxidation reaction connects the CPR-I monomer (Suzhou Gima, Cat#13-2601-XX) to the 5'end of the antisense strand to form 5'-phosphate nucleotides.
  • the deprotection, coupling, capping, and oxidation reaction conditions used to synthesize the sense chain are the same.
  • the steps of cutting and deprotection, purification and desalting are performed, and finally the antisense strand AS is obtained by freeze-drying.
  • siRNA contained in these conjugates are shown in Table 3 and correspond to the conjugates L10-siFXIa1M1SP, L10-siFXIb1M1SP, L10-siFXIc1M1SP, L10-siFXId1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP, L10M1SP, L10M1SP, L10M1SP -The sequence of siFXIi1M1S or L10-siFXIi1M1SP.
  • siRNA sequences listed in Table 4 were synthesized by solid-phase synthesis method, and the molecular weight was detected. Use DEPC water to dissolve the mutually complementary sense strands and antisense strands in equimolar Table 4, and then anneal to obtain the following siRNAs provided by the present disclosure: siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIe1M1SP, siFXIf1M1SP, siFXIeSP, and siXIXIF1M1SP, siFXIeSP, and siHXIFXI1M1SP , As shown in Table 4.
  • the target sequence contains unmodified nucleotides.
  • the target sequence contains unmodified nucleotides.
  • ammonia treatment relative to the amount of single-stranded nucleic acid, use 0.4ml/ ⁇ mol N-methylpyrrolidone to dissolve the product, and then add 0.3ml/ ⁇ mol triethylamine and 0.6ml/ ⁇ mol triethylamine trihydrofluoride to remove 2'-TBDMS protection on ribose.
  • the antisense strand is connected After the last nucleoside monomer in the chain, the CPR-I monomer (Suzhou Gima, Cat#13-2601-XX) is connected to the 5'of the antisense chain through four steps of deprotection, coupling, capping and oxidation. At the end, a 5'-phosphate nucleotide is formed.
  • the deprotection, coupling, capping, and oxidation reaction conditions used are the same as those for the synthesis of the sense strand.
  • the steps of cutting and deprotection, purification and desalting are performed, and finally the antisense strand AS is obtained by freeze-drying.
  • Comparative preparation example 1 Synthesis of reference siRNA
  • the sense strand and antisense strand corresponding to the siRNA numbered NC in Table 4 were synthesized by solid-phase synthesis, and the molecular weight was detected. Use DEPC water to dissolve the obtained equimolar sense strand and antisense strand, and then anneal to obtain the reference siRNA, numbered NC.
  • the capital letters C, G, U, A indicate the base composition of nucleotides;
  • 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 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 of the letter s are connected by phosphorothioate groups;
  • the uppercase letter P indicates the letter The adjacent nucleotide to the right of P is a 5'-phosphate nucleotide.
  • siRNA or conjugate of the present disclosure is prepared, it is lyophilized and stored as a solid powder for later use. When in use, it can be re-dissolved into a solution of the desired concentration using, for example, water for injection, physiological saline (NS), phosphate buffer (PB) or phosphate buffer (PBS).
  • NS physiological saline
  • PB phosphate buffer
  • PBS phosphate buffer
  • DMEM complete medium Hyclone
  • FBS fetal bovine serum
  • penicillin double antibody Penicillin-Streptomycin, Gibco, Invitrogen
  • modified siRNA with a DNA seeded arm is a powerful tool for mammalian gene silence with significantly reduced off-target, effect. 36 Reduced off-search, effect. ), the method described in 2136-2151, construct a detection plasmid, transfect the siRNA to be evaluated (siFXIe1) into HEK293A cells, and reflect the inhibitory activity of siRNA through the expression level of the dual luciferase reporter gene. Specific steps are as follows:
  • the psiCHECK TM -2 (Promega TM ) 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 siFXIe1 (obtained from Preparation Example 20) is:
  • the target sequence was cloned into the Xho I/Not I site of the psiCHECK TM -2 plasmid.
  • Each 1A1 solution contains 1 ⁇ l of 10nM siRNA working solution, 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 3nM, 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.
  • transfection complex 1X1 In the three culture wells, respectively add the transfection complex 1X1, mix 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 1.
  • transfection complex 1X2 In the other three culture wells, add the transfection complex 1X2, mix them evenly, and add 20 ⁇ l/well to obtain a co-transfection mixture with a final siRNA concentration of about 0.03nM, which is recorded as test group 2.
  • the transfection complex 1X3 was added to obtain a transfection mixture without siRNA.
  • the added amount was 20 ⁇ l/well, which was recorded as the control group.
  • each well was supplemented with 100 ⁇ l of H-DMEM complete medium containing 20% FBS. Place the 96-well plate in a CO 2 incubator and continue culturing 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 holes;
  • 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%.
  • Comparative experiment example 1 Reference siRNA NC in vitro (in vitro) inhibitory activity.
  • the inhibitory activity of the reference siRNA NC in the psiCHECK system was also investigated according to the method of Experimental Example 1. The only difference is that the tested siRNA is the reference siRNA NC. The results are shown in Table 5.
  • siFXIe1 has good in vitro inhibitory activity against the target sequence at each concentration, and exhibits a concentration-dependent inhibitory activity.
  • the target sequence inhibition rate was 72.43%, showing a good effect of inhibiting FXI gene expression.
  • This experimental example investigated the IC 50 values of siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIe1M1SP and siFXIi1M1SP in the in vitro psiCHECK system.
  • 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 a detection plasmid, co-transfect the detection plasmid and the test siRNA into HepG2 cells, and reflect the on-target activity and off-target effect of the siRNA through the expression level of the dual luciferase reporter gene . Specific steps are as follows:
  • a detection plasmid was constructed using the psiCHECK TM -2 (Promega TM ) plasmid, which contained a target sequence with the sequence shown in Genbank registration number NM_00128.3.
  • the target sequence was cloned into the Xho I/Not I site of the psiCHECK TM -2 plasmid.
  • HepG2 cells were purchased from Guangzhou Genio Biotech Co., Ltd., and used DMEM containing 20% fetal bovine serum (FBS, HyClone) and 0.2% by volume of penicillin streptomycin (Penicillin-Streptomycin, Gibco, Invitrogen) Cells were cultured in complete medium (HyClone) and cultured in an incubator containing 5% CO 2 /95% air at 37°C.
  • FBS fetal bovine serum
  • penicillin streptomycin Penicillin-Streptomycin, Gibco, Invitrogen
  • siRNAs Use DEPC water to dilute the above detection plasmid into 200ng/ ⁇ l detection plasmid working solution; use DEPC water to prepare each of the following siRNAs into 100nM, 33.3nM, 11.1nM, 3.70nM, 1.23nM, 4.12 There are 10 different concentrations of siRNA working solutions of nM, 0.137nM, 0.0457nM, 0.0152nM and 0.00508nM.
  • the siRNAs used are siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIe1M1SP, and siFXIi1M1SP.
  • Each 2A1-2A10 solution contains 1 ⁇ l of the above 10 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 siRNA transfection complex 2X1-2X10 In the culture well, add each siRNA transfection complex 2X1-2X10, mix evenly, add 20 ⁇ l/well, and get the final concentration of each siRNA to be about 1nM, 0.333nM, 0.111nM, 0.0370nM, 0.0123nM, respectively , 0.00412nM, 0.00137nM, 0.000457nM, 0.000152nM and 0.0000508nM transfection complexes, each siRNA transfection complex 2X1-2X10 was transfected into 3 culture wells respectively to obtain a co-transfection mixture containing siRNA. For the test group.
  • the transfection complex 1X3 was added at a volume of 20 ⁇ l/well to obtain a co-transfection mixture without siRNA, which was recorded as a control group.
  • each well was supplemented with 100 ⁇ l of H-DMEM complete medium containing 20% FBS. Place the 96-well plate in a CO 2 incubator and continue culturing 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 holes;
  • 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.
  • the inhibition rate of siRNA was (1-R) ⁇ 100%.
  • Y is the ratio R, which is the residual activity
  • X is the logarithm of the transfection siRNA concentration
  • Bot is the Y value at the bottom of the steady-state period
  • Top is the Y value at the top of the steady-state period
  • X' is the X value obtained by fitting when Y is halfway from the bottom to the top
  • HillSlope is the slope of the curve obtained by fitting at X'.
  • siRNA number IC 50 Preparation Example 11 siFXIa1M1SP 0.024nM Preparation Example 12 siFXIb1M1SP 0.078nM Preparation Example 13 siFXIc1M1SP 0.119nM Preparation Example 14 siFXId1M1SP 0.071nM Preparation Example 15 siFXIe1M1SP 0.013nM Preparation Example 19 siFXIi1M1SP 0.041nM
  • the siRNA provided by the present disclosure has a high inhibitory activity against the target sequence 1 in HepG2 cells in vitro, with an IC 50 between 0.013-0.119 nM.
  • siRNAs Use DEPC water to formulate each of the following siRNAs into 7 different concentrations of siRNA working solutions of 20 ⁇ M, 6.67 ⁇ M, 2.22 ⁇ M, 0.741 ⁇ M, 0.247 ⁇ M, 0.0823 ⁇ M and 0.0274 ⁇ M.
  • the siRNAs used are respectively siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP or siFXId1M1SP.
  • 3A1-3A7 solution For each siRNA, prepare 3A1-3A7 solution.
  • Each 3A1-3A7 solution contains 3 ⁇ l of the above 7 concentrations of siRNA working solution and 50 ⁇ l of Opti-MEM medium.
  • each 3B solution contains 1 ⁇ l Lipofectamine TM 2000 and 50 ⁇ l Opti-MEM medium.
  • each siRNA transfection complex 3X1-3X7 In the culture well, add each siRNA transfection complex 3X1-3X7, mix evenly, and add 100 ⁇ l/well to obtain the final concentration of each siRNA is about 100nM, 33.3nM, 11.1nM, 3.70nM, 1.23nM, respectively , 0.412nM, 0.137nM transfection mixture, and each siRNA transfection complex 3X1-3X7 was transfected into 3 culture wells respectively to obtain the siRNA-containing transfection mixture, which was recorded as the test group.
  • the transfection complex 3X8 was added, and the addition amount was 100 ⁇ l/well to obtain the transfection mixture without siRNA, which was recorded as the control group.
  • each well was supplemented with 1 ml of H-DMEM complete medium containing 20% FBS. Place the 24-well plate in a CO 2 incubator and continue culturing for 24 hours.
  • RNAVzol purchased from Wiglas Biotechnology (Beijing) Co., Ltd., Catalog No. N002
  • Wiglas Biotechnology Beijing Co., Ltd., Catalog No. N002
  • RNA samples in each well take 1 ⁇ g of total RNA and use the reagents provided by the reverse transcription kit Goldenstar TM RT6 cDNA Synthesis Kit (purchased from Beijing Kinco Xinye Biotechnology Co., Ltd., item number TSK301M), among which Goldenstar TM Oligo (dT ) 17 as the primer, configure 20 ⁇ l of the reverse transcription reaction system according to the reverse transcription operation steps in the kit instructions, and perform reverse transcription on the total RNA of the cell.
  • the conditions for reverse transcription are: incubate the reverse transcription reaction system at 50°C for 50 minutes, then incubate at 85°C for 5 minutes, and finally incubate at 4°C for 30 seconds. After the reaction is over, add 80 ⁇ l of DEPC water to the reverse transcription reaction system to obtain cDNA solution.
  • each reverse transcription reaction system For each reverse transcription reaction system, take 5 ⁇ l of the above cDNA-containing solution as a template, and use SYBR qPCR SuperMix Plus kit (purchased from Nearshore Protein Technology Co., Ltd., catalog number E096-01B) provides a reagent configuration of 20 ⁇ l qPCR reaction system, of which the PCR primer sequences used to amplify the target gene FXI and the internal reference gene GAPDH are shown in Table 7 As shown, the final concentration of each primer is 0.25 ⁇ M. Place each qPCR reaction system on the ABI StepOnePlus Real-Time PCR machine, and use the three-step method for amplification.
  • the amplification program is 95°C pre-denaturation for 10 minutes, then 95°C denaturation for 30s, 60°C annealing for 30s, and 72°C extension for 30s.
  • the product W containing the amplified target gene FXI and the internal reference gene GAPDH is obtained.
  • the product W was incubated at 95°C for 15s, 60°C for 1min, and 95°C for 15s.
  • the real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene FXI and the internal reference gene GAPDH in the product W to obtain the Ct of the target gene FXI and the internal reference gene GAPDH. value.
  • ⁇ Ct (test group) Ct (test group target gene)-Ct (test group internal reference gene)
  • ⁇ Ct (control group) Ct (control group target gene)-Ct (control group internal reference gene)
  • ⁇ Ct (test group) ⁇ Ct (test group)- ⁇ Ct (control group average)
  • ⁇ Ct (control group) ⁇ Ct (control group)- ⁇ Ct (control group average)
  • ⁇ Ct control group average
  • ⁇ Ct control group average
  • control group normalize the FXI mRNA expression level of the test group, and define the FXI mRNA expression level of the control group as 100%.
  • the relative expression level of FXI mRNA in the test group 2- ⁇ Ct (test group) ⁇ 100%.
  • the average relative expression level of the test group FXI mRNA at each concentration is the arithmetic average of the relative expression levels of the three culture wells at that concentration.
  • the log(inhibitor) vs.response—Variable slope dose-response curve was fitted with the non-linear regression analysis function of Graphpad 5.0 software, and the IC 50 value of each siRNA to FXI mRNA was calculated according to the dose-response curve.
  • the dose-effect curve obtained by fitting conforms to the following calculation formula:
  • Y is the relative expression level of FXI mRNA in each test group
  • X is the logarithm of the final concentration of siRNA used in the corresponding test group
  • Bot is the Y value at the bottom of the steady-state period
  • Top is the Y value at the top of the steady-state period
  • X' is the X value obtained by fitting when Y is halfway from the bottom to the top
  • HillSlope is the slope of the curve obtained by fitting at X'.
  • the siRNA provided by the present disclosure has a high activity of inhibiting FXI mRNA in the HepG2 cell line in vitro, with an IC 50 between 1.49-11.1 nM.
  • Experimental example 4 IC 50 detection of FXI mRNA by siRNA in primary mouse liver cells
  • Primary mouse liver cells are extracted from fresh liver tissues of normal C57BL/6N mice. They are inoculated with appropriate density of cells in type I collagen-coated glass or plastic coverslips or tissue culture dishes. The cells were cultured in RPMI 1460 medium with 10% FBS, and cultured in an incubator containing 5% CO 2 /95% air at 37°C for 30 min.
  • the inhibitory activity of siRNA on FXI mRNA and the IC 50 value were measured.
  • the only difference is that the siRNA tested is siFXIf1M1SP; the cells used are mouse liver primary cells; the final concentration of siRNA is 100nM, 25nM, 6.25nM, 1.56nM, 0.391nM, 0.098nM, 0.0244nM and 6.1 ⁇ 10 -3 nM, a total of 8 concentrations.
  • the results are shown in Table 9.
  • siFXIf1M1SP has a high inhibitory activity of FXI mRNA in primary mouse liver cells in vitro, with an IC 50 of 0.021 nM.
  • the inhibition rate of siRNA on FXI mRNA expression was measured.
  • the siRNA used are siFXIg1M1SP and siFXIh1M1SP; for each siRNA, the final concentration of siRNA is 50nM, 5nM, and 0.5nM, a total of 3 2 culture wells for each concentration, the results are shown in Table 10.
  • the siRNA provided by the present disclosure has high inhibitory activity in HepG2 cells in vitro, and can reach an FXI mRNA inhibition rate of 83% when the siRNA concentration is 50 nM.
  • mice C57BL/6N mice were randomly divided into groups (all females), 5 mice in each group, numbered separately.
  • the test conjugates L10-siFXIf1M1S, L10-siFXIi1M1S or L10-siFXIi1M1SP were administered at different doses of 5 mg/kg and 1 mg/kg (both as siRNA) to each group of mice by subcutaneous injection.
  • the siRNA conjugates were provided in the form of 0.9% sodium chloride aqueous solution of 1 mg/ml and 0.2 mg/ml siRNA conjugates respectively, and the administration volume was 5 ml/kg.
  • mice One group of mice was given 1 ⁇ PBS with a volume of 5ml/kg as a control group.
  • RNA was collected separately and stored with RNA later (Sigma Aldrich); the liver tissue was homogenized with a tissue homogenizer, and then Trizol (Thermo Fisher) was used as described in the instructions The operation steps of extracting total RNA.
  • Fluorescence quantitative PCR detection and calculation of FXI mRNA expression level and inhibition rate were carried out according to the method of Experimental Example 3. The only difference is: Use ImProm-IITM Reverse Transcription Kit (Promega) to reverse transcribed the extracted total RNA into cDNA to obtain a cDNA-containing solution, and then use a fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.) to detect the expression level of FXI mRNA in liver tissue.
  • mGAPDH mouse GAPDH
  • FXI and mouse GAPDH are detected using primers for FXI and primers for mouse GAPDH, respectively.
  • control group is the control group mice administered with PBS in the experiment
  • each test group is the administration group mice administered with different siRNA conjugates.
  • the FXI mRNA expression level of the control group was recorded as 100%, and correspondingly, the FXI mRNA expression level inhibition rate was recorded as 0%.
  • the test results were standardized with the FXI mRNA expression level of the control group. The results are shown in Table 12.
  • the siRNA conjugate of the present disclosure shows 56.8-78.4% FXI mRNA inhibition rate at a dose of 1 mg/kg siRNA; at a siRNA concentration of 5 mg/kg, FXI mRNA inhibition rate It is even as high as 95.0%, showing an excellent inhibitory effect on FXI mRNA.
  • the C57BL/6N mice were randomly divided into 7 groups (all male), 5 mice in each group, respectively numbered.
  • the conjugates L10-siFXIf1M1S and L10-siFXIi1M1SP were respectively administered to each of the three groups of mice.
  • One group of mice was given saline (Saline) as a control group.
  • the administration method adopts the subcutaneous administration method.
  • the conjugate is provided in the form of 0.9% sodium chloride aqueous solution of 1.8 mg/ml (calculated as siRNA) siRNA conjugate, and the physiological saline is 0.9% sodium chloride aqueous solution.
  • the volume is 5ml/k, and the dosage of the conjugate is 9mg/kg.
  • the animals in the administration group were sacrificed on the 29th day after administration, and the animals in the control group were sacrificed on the 8th day after administration.
  • the liver of each mouse was collected separately, stored in RNA later (Sigma Aldrich), and the liver tissue was homogenized with a tissue homogenizer, and then Trizol (Thermo Fisher) was used to extract total RNA according to the operation steps described in the instructions.
  • the FXI mRNA expression level of each mouse was detected by fluorescent quantitative PCR and the FXI mRNA expression level and inhibition rate were calculated.
  • the only difference is: the use of ImProm-IITM reverse transcription kit (Promega) According to its instructions, the extracted total RNA was reverse transcribed into cDNA to obtain a cDNA-containing solution, and then a fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.) was used to detect the expression level of FXI mRNA in liver tissue.
  • mouse GAPDH (mGAPDH) gene is used as an internal reference gene, and FXI and mouse GAPDH are detected using primers for FXI and primers for mouse GAPDH, respectively. See Table 11 for the sequence of the detection primer.
  • the control group is the control group mice that were administered saline in the experiment. Each test group was administered with different siRNA conjugates and sampled after different administration times. Medicine group mice. The FXI mRNA expression level of the control group was recorded as 100%, and correspondingly, the FXI mRNA expression level inhibition rate was recorded as 0%. The test results were standardized with the FXI mRNA expression level of the control group. The results are shown in Table 13. In Table 13, the FXI mRNA expression level inhibition rate is the arithmetic mean of the FXI mRNA expression level inhibition rate of 5 mice in the group after the corresponding number of days after administration of the corresponding siRNA conjugate.
  • the siRNA conjugate of the present disclosure showed excellent FXI mRNA inhibition rate in the liver at different time points over a long period of time, with an inhibition rate of at least 89.18% , Even as high as 92.89%.
  • mice all showed a significantly prolonged APTT detection value for a longer period of time, with a maximum prolongation of 64.9%. It can be seen that the siRNA conjugate of the present disclosure can effectively prolong the clotting time of mice, indicating that it has excellent application prospects for the treatment and/or prevention of thrombotic diseases and ischemic stroke.
  • mice used in this experiment were purchased from Saiye Biotechnology Co., Ltd. The humanized mice were randomly divided into groups of 4 (2 males and 2 females), and the conjugates were given to each group of mice.
  • Each conjugate was administered in the form of a 0.9% sodium chloride aqueous solution of 0.3 mg/ml (calculated as siRNA), and the administration volume was 10 ml/kg mice Body weight, that is, the dosage of each conjugate is 3 mg/kg (calculated as siRNA).
  • siRNA 0.9% sodium chloride aqueous solution of 0.3 mg/ml
  • the administration volume was 10 ml/kg mice Body weight, that is, the dosage of each conjugate is 3 mg/kg (calculated as siRNA).
  • siRNA sodium citrate dihydrate aqueous solution was added at the ratio of anticoagulant to plasma volume ratio of 1:9 (v/v) to prevent Blood coagulation and centrifugation to separate plasma.
  • RNA later Take about 100mg/mouse of the left lobe of the liver and save it with RNA later (Sigma Aldrich); then for each mouse’s liver tissue, homogenize the liver tissue with a tissue homogenizer, and then use Trizol (Thermo Fisher) according to The operation steps described in the instructions extract the total RNA from the liver tissue of each mouse.
  • the FXI mRNA expression level of the control group is recorded as 100%.
  • the FXI mRNA expression level inhibition rate is recorded as 0%, and the test result is based on the control group
  • the FXI mRNA expression levels of FXI were normalized, and the results are shown in Table 16.
  • the human FXI mRNA inhibition rate is the average value and its standard deviation of a group of mouse human FXI mRNA inhibition rates given the corresponding siRNA conjugate.
  • Table 16 Inhibition rate of siRNA conjugates of the present disclosure on human FXI mRNA in humanized mice
  • the siRNA of the present disclosure shows a good inhibitory effect on human FXI mRNA in the liver of humanized heterozygous mice, and the inhibition rate on FXI mRNA is as high as about 71-93%.
  • mice administered conjugates L10-siFXIf1M1S, L10-siFXIa1M1SP, L10-siFXIb1M1SP, L10- The FXI protein content in plasma obtained from the test group of siFXIc1M1SP, L10-siFXId1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP, L10-siFXIh1M1SP or L10-siFXIi1M1S and the saline control group.
  • 108 ⁇ L of the diluted sample diluent was added to 12 ⁇ L of plasma to obtain 10-fold diluted plasma.
  • Dissolve the FXI detection antibody in the kit (labeled ItemF in the kit) with 100 ⁇ L of the diluted sample diluent as an antibody sample, then take 75 ⁇ L of this antibody sample and add 5925 ⁇ L of the diluted sample diluent to 80-fold dilution to obtain the test Antibody solution.
  • the washing buffer in the kit (labeled ItemB in the kit) was diluted 20 times with deionized water to obtain a diluted washing solution.
  • Standard concentration gradient solutions one of which is the diluted sample diluent (can be regarded as 0pg/mL standard solution), and the other 7 are the standard products in the kit (marked as ItemC in the kit) )
  • Y is the corresponding optical density value read at 450nm
  • X is the log value of the concentration in the standard curve ( ⁇ g/mL)
  • Bot is the Y value at the bottom of the steady-state period
  • Top is the Y value at the top of the steady-state period
  • X' is the X value obtained by fitting when Y is halfway from the bottom to the top
  • HillSlope is the slope of the curve at X'
  • the FXI protein inhibition rate (control group protein concentration-test group protein concentration)/control group protein concentration ⁇ 100%.
  • concentration results and inhibition rate data are shown in Table 17.
  • the FXI protein concentration and the FXI protein inhibition rate are respectively the arithmetic mean of the FXI protein concentration and FXI protein inhibition rate of a group of mice given the corresponding siRNA conjugate.
  • Table 17 Inhibition of the siRNA conjugate of the present disclosure on the expression effect of plasma protein concentration
  • the siRNA conjugates of the present disclosure all show very excellent effects of inhibiting the expression of human FXI protein in humanized heterozygous mouse plasma, especially the conjugates L10-siFXIa1M1SP and L10- Both siFXIg1M1SP showed a high FXI protein inhibition rate of about 99%.

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Abstract

提供了抑制血浆凝血因子XI基因表达的siRNA，含有siRNA的药物组合物和缀合物，试剂盒，以及siRNA及其药物组合物和缀合物在制备用于治疗和/或预防血栓性疾病和缺血性卒中的药物中的用途。

Description

核酸、药物组合物与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血浆凝血因子XI(Factor XI，FXI)基因表达的核酸和含有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
血浆凝血因子XI(Factor XI，以下简称为FXI)是接触激活途径(contact activation pathway)的关键组分，有助于凝血酶的产生，而凝血酶又是参与纤维蛋白的形成和纤维蛋白溶解的保护的重要组分。高FXI水平是静脉血栓形成的危险因素。通过抑制FXI基因的表达，能够在细胞水平上对血栓性疾病、特别是静脉血栓以及缺血性卒中进行预防和治疗。
小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNA interference，RNAi)这一机制，以序列特异性的方式抑制或阻断任何感兴趣的目的基因的表达，从而达到治疗疾病的目的。
开发抑制FXI基因表达和治疗血栓性疾病的siRNA药物的关键之一在于寻找合适的siRNA及其修饰以及有效的递送系统。
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FXI基因的表达，含有siRNA的药物组合物或siRNA缀合物能够特异性地靶向肝脏，从而可以抑制肝脏中FXI基因的表达，实现血栓性疾病的治疗或预防，从而完成了本发明。
在一些实施方案中，本公开提供了第一种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，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'-GGGUAUUCUUUCAAGCAAZ 1-3'(SEQ ID NO:1)；
5'-Z 2UUGCUUGAAAGAAUACCC-3'(SEQ ID NO:2)，
其中，Z 1为U，Z 2为A，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。
在一些实施方案中，本公开提供了第二种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO: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'-GGCAUAAACUAUAACAGCZ 5-3'(SEQ ID NO:61)；
5'-Z 6GCUGUUAUAGUUUAUGCC-3'(SEQ ID NO:62)，
其中，Z 5为U，Z 6为A，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。
在一些实施方案中，本公开提供了第三种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO: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'-GCUCAAGAAUGCCAAGAAZ 9-3'(SEQ ID NO:121)；
5'-Z 10UUCUUGGCAUUCUUGAGC-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。
在一些实施方案中，本公开提供了第四种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO: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'-GCAACAAAGACAUUUAUGZ 13-3'(SEQ ID NO:181)；
5'-Z 14CAUAAAUGUCUUUGUUGC-3'(SEQ ID NO:182)，
其中，Z 13为U，Z 14为A，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。
在一些实施方案中，本公开提供了第五种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO: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'-GAAUCUCAAAGAAAUCUUZ 17-3'(SEQ ID NO:241)；
5'-Z 18AAGAUUUCUUUGAGAUUC-3'(SEQ ID NO:242)，
其中，Z 17为U，Z 18为A，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。
在一些实施方案中，本公开提供了第六种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO: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'-GUACGUGGACUGGAUUCUZ 21-3'(SEQ ID NO:301)；
5'-Z 22AGAAUCCAGUCCACGUAC-3'(SEQ ID NO:302)，
其中，Z 21为G，Z 22为C，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。
在一些实施方案中，本公开提供了第七种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO: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'-AUUUCUGGGUAUUCUUUCZ 25-3'(SEQ ID NO:361)；
5'-Z 26GAAAGAAUACCCAGAAAU-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。
在一些实施方案中，本公开提供了第八种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO: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'-CAUGAAGGGCAUAAACUAZ 29-3'(SEQ ID NO:421)；
5'-Z 30UAGUUUAUGCCCUUCAUG-3'(SEQ ID NO:422)，
其中，Z 29为U，Z 30为A，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。
在一些实施方案中，本公开提供了第九种能够抑制FXI基因表达的siRNA，该siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:481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:482所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GGAUUCUGGAGAAAACUCZ 33-3'(SEQ ID NO:481)；
5'-Z 34GAGUUUUCUCCAGAAUCC-3'(SEQ ID NO:482)，
其中，Z 33为A，Z 34为U，所述核苷酸序列I中包含位置对应于Z 33的核苷酸Z 35，所述核苷酸序列II中包含位置对应于Z 34的核苷酸Z 36，所述Z 36是所述反义链5'末端的第一个核苷酸。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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FXI基因异常表达引起的血栓性疾病和/或缺血性卒中的药物中的用途。
在一些实施方案中，本公开提供了一种治疗和/或预防血栓性疾病和/或缺血性卒中的方法，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缀合物给予患有血栓性疾病和/或缺血性卒中的受试者。
在一些实施方案中，本公开提供了一种抑制肝细胞中FXI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缀合物与所述肝细胞接触。
在一些实施方案中，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。
有益效果
本公开提供的siRNA、药物组合物和siRNA缀合物具有良好的稳定性，较高的FXI mRNA抑制活性，较低的脱靶效应，和/或能显著治疗或缓解血栓性疾病和/或缺血性卒中症状。
在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物在体外细胞实验中显示出优异的靶基因抑制活性。在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物在肝细胞中显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的靶基因表达抑制率。在一些实施方案中，本公开提供的siRNA在psiCHECK系统中对FXI mRNA的IC 50的抑制活性，对FXI mRNA的IC 50在0.013-0.119nM之间。在一些实施方案中，本公开提供的siRNA在HepG2细胞中显示出很高的抑制活性，对FXI mRNA的IC 50在1.49-11.1nM之间。在一些实施方案中，本公开提供的siRNA缀合物在小鼠肝原代细胞中显示出很高的抑制活性，对FXI mRNA的IC 50在0.012-3.86nM之间。在一些实施方案中，本公开提供的siRNA在HepG2细胞中对FXI mRNA表达量的抑制在浓度为50nM时可高达86.9％的FXI mRNA抑制率。
在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物可在体内具有更高的稳定性和/或更高的活性。在一些实施方案中，本公开提供的siRNA、药物组合物或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。在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的FXI基因表达抑制率。在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的肝内FXI基因表达抑制率。在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的动物模型中肝内FXI基因表达抑制率。在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物在体内显示出至少20％、30％、40％、50％、60％、70％、80％、90％或95％的人类受试者中肝内FXI基因表达抑制率。在一些实施方案中，本公开提供的siRNA缀合物在小鼠体内对FXI mRNA表达的抑制在5mg/kg的siRNA浓度下，抑制率可达95.0％。在一些实施方案中，本公开提供的siRNA缀合物在人源化小鼠体内对人FXI mRNA表达的抑制在3mg/kg的siRNA浓度下，抑制率可达93.09％，同时可显著抑制血浆FXI蛋白浓度，抑制率可达99％左右。在一些实施方案中，本公开提供的siRNA缀合物在CD57小鼠体内可显著延长血浆APTT检测值，例如，可延长64.9％。
在一些实施方案中，本公开提供的siRNA、药物组合物或siRNA缀合物未显示出明显脱靶效应。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。
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FXI基因的表达，有效治疗和/或预防由FXI基因过量表达引起的血栓性疾病和/或缺血性卒中症状，具有良好的应用前景。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
在本公开中，FXI mRNA是指具有Genbank注册号为NM_000128.3所示序列的mRNA。进一步地，若无其它说明，本公开中所使用的术语“靶基因”是指转录上述FXI mRNA的基因，术语“靶mRNA”是指上述FXI mRNA。
定义
在上文及下文中，如无特别说明，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相 邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；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(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。
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。在一些实施方案中，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的药物组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(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siRNA缀合物的总称、式(305)和式(307)所示的siRNA缀合物总称，或式(305)、式(307)、式(308)所示的siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开的siRNA缀合物的特定化合物。
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，C 1-C 6烷基包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1,3-二烯-1-基、丁-1,3-二烯-2-基等等。在某些实施方案中，烯基基团具有2到20个碳原子，而在其他实施方案中，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。在某些实施方案中，炔基具有2到20个碳原子，而在其他实施方案中，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“卤素”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-氟乙基或五氟乙基。
“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、氧或硫。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连接至分子的其余部分。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团对特定的反应条件不敏感，并且可以在分子中的该官能团上添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2,2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献各自整体并入本文。在一些实施方案中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方案中，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)或9-(对甲氧基苯基)氧杂蒽-9-基(Mox)。在一些实施方案中，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)或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、大鼠或任何种类的家禽。
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九种能够抑制FXI基因表达的siRNA。以下依次对其进行详细描述。
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。
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'-GGGUAUUCUUUCAAGCAAZ 1-3'(SEQ ID NO:1)；
5'-Z 2UUGCUUGAAAGAAUACCC-3'(SEQ ID NO:2)，
其中，Z 1为U，Z 2为A，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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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U、C或G。在一些实施方案中，所述核苷酸差异为Z 4位置处的差异，Z 4选自U、C或G。在一些实施方案中，Z 3是与Z 4互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有碱基错配。
在一些实施方案中，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'-GGGUAUUCUUUCAAGCAAZ 3-3'(SEQ ID NO:3)；
5'-Z 4UUGCUUGAAAGAAUACCC-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；在一些实施方案中，Z 3为U，Z 4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。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。在一些实施方案中，所述siRNA正义链和反义链的长度比为19/21、21/23或23/25。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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。在一些实施方案中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:1表示的核苷酸序列的5'末端相邻，且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方案中，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CU，核苷酸序列IV的碱基组成为AG；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UCU，核苷酸序列IV的碱基组成为AGA；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UUCU，核苷酸序列IV的碱基组成为AGAA；此时，正义链和反义链的长度比为23/23。在一些实施方案中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU，核苷酸序列IV的碱基组成为AG；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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'-GGCAUAAACUAUAACAGCZ 5-3'(SEQ ID NO:61)；
5'-Z 6GCUGUUAUAGUUUAUGCC-3'(SEQ ID NO:62)，
其中，Z 5为U，Z 6为A，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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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U、C或G。在一些实施方案中，所述核苷酸差异为Z 8位置处的差异，Z 8选自U、C或G。在一些实施方案中，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mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，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'-GGCAUAAACUAUAACAGCZ 7-3'(SEQ ID NO:63)；
5'-Z 8GCUGUUAUAGUUUAUGCC-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；在一些实施方案中，Z 7为U，Z 8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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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mRNA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。
在一些实施方案中，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AG，核苷酸序列IV的碱基组成为CU；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AAG，核苷酸序列IV的碱基组成为CUU；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GAAG，核苷酸序列IV的碱基组成为CUUC；此时，正义链和反义链的长度比为23/23。在一些实施方案中，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AG，核苷酸序列IV的碱基组成为CU；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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'-GCUCAAGAAUGCCAAGAAZ 9-3'(SEQ ID NO:121)；
5'-Z 10UUCUUGGCAUUCUUGAGC-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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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。在一些实施方案中，所述核苷酸差异为Z 12位置处的差异，Z 12选自A、C或G。在一些实施方案中，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mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，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'-GCUCAAGAAUGCCAAGAAZ 11-3'(SEQ ID NO:123)；
5'-Z 12UUCUUGGCAUUCUUGAGC-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；在一些实施方案中，Z 11为A，Z 12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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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mRNA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。
在一些实施方案中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为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AGU，核苷酸序列IV的碱基组成为ACU；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GAGU，核苷酸序列IV的碱基组成为ACUC；此时，正义链和反义链的长度比为23/23。在一些实施方案中，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。
在一些实施方案中，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'-GCAACAAAGACAUUUAUGZ 13-3'(SEQ ID NO:181)；
5'-Z 14CAUAAAUGUCUUUGUUGC-3'(SEQ ID NO:182)，
其中，Z 13为U，Z 14为A，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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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U、C或G。在一些实施方案中，所述核苷酸差异为Z 16位置处的差异，Z 16选自U、C或G。在一些实施方案中，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mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，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'-GCAACAAAGACAUUUAUGZ 15-3'(SEQ ID NO:183)；
5'-Z 16CAUAAAUGUCUUUGUUGC-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；在一些实施方案中，Z 15为U，Z 16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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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mRNA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。
在一些实施方案中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A；此时，正义链和反义链的长 度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UU，核苷酸序列IV的碱基组成为AA；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CUU，核苷酸序列IV的碱基组成为AAG；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GCUU，核苷酸序列IV的碱基组成为AAGC；此时，正义链和反义链的长度比为23/23。在一些实施方案中，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UU，核苷酸序列IV的碱基组成为AA；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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'-GAAUCUCAAAGAAAUCUUZ 17-3'(SEQ ID NO:241)；
5'-Z 18AAGAUUUCUUUGAGAUUC-3'(SEQ ID NO:242)，
其中，Z 17为U，Z 18为A，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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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U、C或G。在一些实施方案中，所述核苷酸差异为Z 20位置处的差异，Z 20选自U、C或G。在一些实施方案中，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mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，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'-GAAUCUCAAAGAAAUCUUZ 19-3'(SEQ ID NO:243)；
5'-Z 20AAGAUUUCUUUGAGAUUC-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；在一些实施方案中，Z 19为U，Z 20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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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mRNA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。
在一些实施方案中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为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AA，核苷酸序列IV的碱基组成为UU；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AAA，核苷酸序列IV的碱基组成为UUU；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CAAA，核苷酸序列IV的碱基组成为UUUG；此时， 正义链和反义链的长度比为23/23。在一些实施方案中，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AA，核苷酸序列IV的碱基组成为UU；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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'-GUACGUGGACUGGAUUCUZ 21-3'(SEQ ID NO:301)；
5'-Z 22AGAAUCCAGUCCACGUAC-3'(SEQ ID NO:302)，
其中，Z 21为G，Z 22为C，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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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U、G或A。在一些实施方案中，所述核苷酸差异为Z 24位置处的差异，Z 24选自U、G或A。在一些实施方案中，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mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，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'-GUACGUGGACUGGAUUCUZ 23-3'(SEQ ID NO:303)；
5'-Z 24AGAAUCCAGUCCACGUAC-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；在一些实施方案中，Z 23为G，Z 24为C；
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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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mRNA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。
在一些实施方案中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为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GA，核苷酸序列IV的碱基组成为UC；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CGA，核苷酸序列IV的碱基组成为UCG；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UCGA，核苷酸序列IV的碱基组成为UCGA；此时，正义链和反义链的长度比为23/23。在一些实施方案中，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GA，核苷酸序列IV的碱基组成为UC；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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，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: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'-AUUUCUGGGUAUUCUUUCZ 25-3'(SEQ ID NO:361)；
5'-Z 26GAAAGAAUACCCAGAAAU-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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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 4选自A、C或G。在一些实施方案中，所述核苷酸差异为Z 28位置处的差异，Z 4选自A、C或G。在一些实施方案中，Z 27是与Z 28互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，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'-AUUUCUGGGUAUUCUUUCZ 27-3'(SEQ ID NO:363)；
5'-Z 28GAAAGAAUACCCAGAAAU-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，Z 28是与Z 27互补的核苷酸；在一些实施方案中，Z 27为A，Z 28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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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。在一些实施方案中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO: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。
在一些实施方案中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为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CG，核苷酸序列IV的碱基组成为CG；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GCG，核苷酸序列IV的碱基组成为CGC；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AGCG，核苷酸序列IV的碱基组成为CGCU；此时，正义链和反义链的长度比为23/23。在一些实施方案中，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CG，核苷酸序列IV的碱基组成为CG；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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'-CAUGAAGGGCAUAAACUAZ 29-3'(SEQ ID NO:421)；
5'-Z 30UAGUUUAUGCCCUUCAUG-3'(SEQ ID NO:422)，
其中，Z 29为U，Z 30为A，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。
在一些实施方案中，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。
在一些实施方案中，所述核苷酸序列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。
在一些实施方案中，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U、C或G。在一些实施方案中，所述核苷酸差异为Z 32位置处的差异，Z 32选自U、C或G。在一些实施方案中，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mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，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'-CAUGAAGGGCAUAAACUAZ 31-3'(SEQ ID NO:423)；
5'-Z 32UAGUUUAUGCCCUUCAUG-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，Z 32是与Z 31互补的核苷酸；在一些实施方案中，Z 31为U，Z 32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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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。在一些实施方案中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO: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。
在一些实施方案中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为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GA，核苷酸序列IV的碱基组成为UC；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AGA，核苷酸序列IV的碱基组成为UCU；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UAGA，核苷酸序列IV的碱基组成为UCUA；此时，正义链和反义链的长度比为23/23。在一些实施方案中，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GA，核苷酸序列IV的碱基组成为UC；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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。
第九种siRNA
按照本公开，所述siRNA可以是第九种siRNA。
所述第九种siRNA含有正义链和反义链，所述第九种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:481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:482所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GGAUUCUGGAGAAAACUCZ 33-3'(SEQ ID NO:481)；
5'-Z 34GAGUUUUCUCCAGAAUCC-3'(SEQ ID NO:482)，
其中，Z 33为A，Z 34为U，所述核苷酸序列I中包含位置对应于Z 33的核苷酸Z 35，所述核苷酸序列II中包含位置对应于Z 34的核苷酸Z 36，所述Z 36是所述反义链5'末端的第一个核苷酸。
在一些实施方案中，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。
在一些实施方案中，所述核苷酸序列I与SEQ ID NO:481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。
在一些实施方案中，所述核苷酸序列II与SEQ ID NO:482所示的核苷酸序列之间的核苷酸差异包括Z 36位置处的差异，且Z 36选自A、C或G。在一些实施方案中，所述核苷酸差异为Z 36位置处的差异，Z 36选自A、C或G。在一些实施方案中，Z 35是与Z 36互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方案中，核苷酸序列I是SEQ ID NO:483所示的核苷酸序列，核苷酸序列II是SEQ ID NO:484所示的核苷酸序列：
5'-GGAUUCUGGAGAAAACUCZ 35-3'(SEQ ID NO:483)；
5'-Z 36GAGUUUUCUCCAGAAUCC-3'(SEQ ID NO:484)，
其中，所述Z 36是反义链5'末端的第一个核苷酸，Z 35选自A、U、G或C，并且Z 36是与Z 35互补的核苷酸；在一些实施方案中，Z 35为A，Z 36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。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV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。在一些实施方案中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:481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。
在一些实施方案中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU，核苷酸序列IV的碱基组成为AG；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为ACU，核苷酸序列IV的碱基组成为AGU；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GACU，核苷酸序列IV的碱基组成为AGUC；此时，正义链和反义链的长度比为23/23。在一些实施方案中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU，核苷酸序列IV的碱基组成为AG；此时，正义链和反义链的长度比为21/21。
在一些实施方案中，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。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至第九种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、第八种siRNA和第九种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、第八种siRNA或第九种siRNA还含有核苷酸序列V”。
在一些实施方案中，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。在一些实施方案中，所述核苷酸序列V的长度为2个核苷酸，由此，本公开提供的siRNA正义链和反义链的长度比可以是19/21、21/23或23/25。
所述核苷酸序列V中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约合成成本，所述核苷酸序列V为连续的2个胸腺嘧啶脱氧核糖核苷酸(dTdT)或连续的2个尿嘧啶核糖核苷酸(UU)；或者，为了提高siRNA反义链与靶mRNA的亲和力，核苷酸序列V与靶mRNA的相应位置的核苷酸互补。因此，在一些实施方案中，本公开的siRNA的正义链和反义链的长度之比为19/21或21/23，此时，本公开的siRNA具有更好的靶mRNA沉默活性。
靶mRNA的相应位置的核苷酸是指与靶mRNA的一段核苷酸序列在5'末端相邻的核苷酸或核苷酸序列。该段靶mRNA的核苷酸序列是与核苷酸序列II实质上反向互补或完全反向互补，或者与核苷酸序列II和核苷酸序列IV构成的核苷酸序列实质上反向互补或完全反向互补的那段 核苷酸序列。
在一些实施方案中，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'-GGGUAUUCUUUCAAGCAAZ 3-3'(SEQ ID NO:5)；
5'-Z 4UUGCUUGAAAGAAUACCCAG-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'-CUGGGUAUUCUUUCAAGCAAZ 3-3'(SEQ ID NO:7)；
5'-Z 4UUGCUUGAAAGAAUACCCAGAA-3'(SEQ ID NO:8)；
其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C，并且Z 4是与Z 3互补的核苷酸。
在一些实施方案中，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'-GGCAUAAACUAUAACAGCZ 7-3'(SEQ ID NO:65)；
5'-Z 8GCUGUUAUAGUUUAUGCCCU-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'-AGGGCAUAAACUAUAACAGCZ 7-3'(SEQ ID NO:67)；
5'-Z 8GCUGUUAUAGUUUAUGCCCUUC-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。
在一些实施方案中，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'-GCUCAAGAAUGCCAAGAA Z 11-3'(SEQ ID NO:125)；
5'-Z 12UUCUUGGCAUUCUUGAGCAC-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'-GUGCUCAAGAAUGCCAAGAAZ 11-3'(SEQ ID NO:127)；
5'-Z 12UUCUUGGCAUUCUUGAGCACUC-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。
在一些实施方案中，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'-GCAACAAAGACAUUUAUGZ 15-3'(SEQ ID NO:185)；
5'-Z 16CAUAAAUGUCUUUGUUGCAA-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'-UUGCAACAAAGACAUUUAUGZ 15-3'(SEQ ID NO:187)；
5'-Z 16CAUAAAUGUCUUUGUUGCAAGC-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。
在一些实施方案中，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'-GAAUCUCAAAGAAAUCUUZ 19-3'(SEQ ID NO:245)；
5'-Z 20AAGAUUUCUUUGAGAUUCUU-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'-AAGAAUCUCAAAGAAAUCUUZ 19-3'(SEQ ID NO:247)；
5'-Z 20AAGAUUUCUUUGAGAUUCUUUG-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 19选自A、U、G或C，并且Z 20是与Z 19互补的核苷酸。
在一些实施方案中，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'-GUACGUGGACUGGAUUCUZ 23-3'(SEQ ID NO:305)；
5'-Z 24AGAAUCCAGUCCACGUACUC-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'-GAGUACGUGGACUGGAUUCUZ 23-3'(SEQ ID NO:307)；
5'-Z 24AGAAUCCAGUCCACGUACUCGA-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。
在一些实施方案中，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'-AUUUCUGGGUAUUCUUUCZ 27-3'(SEQ ID NO:365)；
5'-Z 28GAAAGAAUACCCAGAAAUCG-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'-CGAUUUCUGGGUAUUCUUUCZ 27-3'(SEQ ID NO:367)；
5'-Z 28GAAAGAAUACCCAGAAAUCGCU-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 27互补的核苷酸。
在一些实施方案中，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'-CAUGAAGGGCAUAAACUAZ 31-3'(SEQ ID NO:425)；
5'-Z 32UAGUUUAUGCCCUUCAUGUC-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'-GACAUGAAGGGCAUAAACUAZ 31-3'(SEQ ID NO:427)；
5'-Z 32UAGUUUAUGCCCUUCAUGUCUA-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。
在一些实施方案中，对于所述第九种siRNA，所述siRNA的正义链含有如SEQ ID NO:485所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:486所示的核苷酸序列：
5'-GGAUUCUGGAGAAAACUCZ 35-3'(SEQ ID NO:485)；
5'-Z 36GAGUUUUCUCCAGAAUCCAG-3'(SEQ ID NO:486)；
或者，所述siRNA的正义链含有如SEQ ID NO:487所示的核苷酸序列，所述反义链含有如SEQ ID NO:488所示的核苷酸序列：
5'-CUGGAUUCUGGAGAAAACUCZ 35-3'(SEQ ID NO:487)；
5'-Z 36GAGUUUUCUCCAGAAUCCAGUC-3'(SEQ ID NO:488)；
其中，所述Z 36是反义链5'末端的第一个核苷酸，Z 35选自A、U、G或C，并且Z 36是与Z 35互补的核苷酸。
在一些实施方案中，本公开所述siRNA为表1a-表1i中列出的siFXIa1、siFXIa2、siFXIb1、siFXIb2、siFXIc1、siFXIc2、siFXId1、siFXId2、siFXIe1、siFXIe2、siFXIf1、siFXIf2、siFXIg1、siFXIg2、siFXIh1、siFXIh2siFXIi1或siFXIi2。
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。在一些实施方案中，本公开的siRNA中的每个核苷酸均为未经修饰的核苷酸。在一些实施方案中，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FXI基因表达的功能明显削弱或丧失。
在一些实施方案中，本公开的siRNA至少含有1个修饰的核苷酸。在本公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基2'位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者具有经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008,13(19-20):842-55中公开的修饰的核苷酸。
在一些实施方案中，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。
在一些实施方案中，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。在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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。在一些实施方案中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
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。
在一些实施方案中，2'-烷氧基修饰的核苷酸为2'-甲氧基(2'-OMe)修饰的核苷酸，如式(8)所示。在一些实施方案中，2'-经取代的烷氧基修饰的核苷酸，例如可以是2'-甲氧基乙基(2'-MOE)修饰的核苷酸，如式(9)所示。在一些实施方案中，2'-氨基(2'-NH 2)修饰的核苷酸如式(10)所示。在一些实施方案中，2'-脱氧核苷酸(DNA)如式(11)所示：
Figure PCTCN2020091484-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。在一些实施方案中，核苷酸类似物可以是异核苷酸、桥联的核苷酸或无环核苷酸。
桥联的核苷酸(bridged nucleic acid，简称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。在一些实施方案中，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 PCTCN2020091484-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。在一些实施方案中，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 PCTCN2020091484-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。在一些实施方案中，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 PCTCN2020091484-appb-000004
上述式(17)-式(18)所示化合物中，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。
在一些实施方案中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，本公开提供的siRNA为表1a-表1i中列出的siFXIa1-M1、siFXIa1-M2、siFXIa1-M3、siFXIa2-M1、siFXIa2-M2、siFXIa2-M3、siFXIb1-M1、siFXIb1-M2、siFXIb1-M3、siFXIb2-M1、siFXIb2-M2、siFXIb2-M3、siFXIc1-M1、siFXIc1-M2、siFXIc1-M3、siFXIc2-M1、siFXIc2-M2、siFXIc2-M3、siFXId1-M1、siFXId1-M2、siFXId1-M3、siFXId2-M1、siFXId2-M2、siFXId2-M3、siFXIe1-M1、siFXIe1-M2、siFXIe1-M3、siFXIe2-M1、siFXIe2-M2、siFXIe2-M3、siFXIf1-M1、siFXIf1-M2、siFXIf1-M3、siFXIf2-M1、siFXIf2-M2、siFXIf2-M3、siFXIg1-M1、siFXIg1-M2、siFXIg1-M3、siFXIg2-M1、siFXIg2-M2、siFXIg2-M3、siFXIh1-M1、siFXIh1-M2、siFXIh1-M3、siFXIh2-M1、siFXIh2-M2、siFXIh2-M3、siFXIi1-M1、siFXIi1-M2、siFXIi1-M3、 siFXIi2-M1、siFXIi2-M2、siFXIi2-M3中的任意一种。
具有上述修饰的siRNA不仅成本低，而且可使血液中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。同时，上述修饰的siRNA具有较高的抑制靶mRNA的活性。
在一些实施方案中，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。在一些实施方案中，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；在一些实施方案中，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 PCTCN2020091484-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。
在一些实施方案中，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。在一些实施方案中，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。在一些实施方案中，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。在一些实施方案中，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。
在一些实施方案中，本公开提供的siRNA为表1a-表1i中列出的siFXIa1-M1S、siFXIa1-M2S、siFXIa1-M3S、siFXIa2-M1S、siFXIa2-M2S、siFXIa2-M3S、siFXIb1-M1S、siFXIb1-M2S、siFXIb1-M3S、siFXIb2-M1S、siFXIb2-M2S、siFXIb2-M3S、siFXIc1-M1S、siFXIc1-M2S、siFXIc1-M3S、siFXIc2-M1S、siFXIc2-M2S、siFXIc2-M3S、siFXId1-M1S、siFXId1-M2S、siFXId1-M3S、siFXId2-M1S、siFXId2-M2S、siFXId2-M3S、siFXIe1-M1S、siFXIe1-M2S、siFXIe1-M3S、siFXIe2-M1S、siFXIe2-M2S、siFXIe2-M3S、siFXIf1-M1S、siFXIf1-M2S、siFXIf1-M3S、siFXIf2-M1S、siFXIf2-M2S、siFXIf2-M3S、siFXIg1-M1S、siFXIg1-M2S、siFXIg1-M3S、siFXIg2-M1S、siFXIg2-M2S、siFXIg2-M3S、siFXIh1-M1S、siFXIh1-M2S、siFXIh1-M3S、siFXIh2-M1S、siFXIh2-M2S、siFXIh2-M3S、FXIi1-M1S、siFXIi1-M2S、siFXIi1-M3S、siFXIi2-M1S、siFXIi2-M2S、siFXIi2-M3S中的任意一种。
在一些实施方案中，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 PCTCN2020091484-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 PCTCN2020091484-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。
在一些实施方案中，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。
在一些实施方案中，本公开提供的siRNA为表1a-表1i中列出的siFXIa1-M1P1、siFXIa1-M2P1、siFXIa1-M3P1、siFXIa2-M1P1、siFXIa2-M2P1、siFXIa2-M3P1、siFXIa1-M1SP1、siFXIa1-M2SP1、siFXIa1-M3SP1、siFXIa2-M1SP1、siFXIa2-M2SP1、siFXIa2-M3SP1、siFXIb1-M1P1、siFXIb1-M2P1、siFXIb1-M3P1、siFXIb2-M1P1、siFXIb2-M2P1、siFXIb2-M3P1、siFXIb1-M1SP1、siFXIb1-M2SP1、siFXIb1-M3SP1、siFXIb2-M1SP1、siFXIb2-M2SP1、siFXIb2-M3SP1、siFXIc1-M1P1、siFXIc1-M2P1、siFXIc1-M3P1、siFXIc2-M1P1、siFXIc2-M2P1、siFXIc2-M3P1、siFXIc1-M1SP1、siFXIc1-M2SP1、siFXIc1-M3SP1、siFXIc2-M1SP1、siFXIc2-M2SP1、siFXIc2-M3SP1、siFXId1-M1P1、siFXId1-M2P1、siFXId1-M3P1、siFXId2-M1P1、siFXId2-M2P1、siFXId2-M3P1、siFXId1-M1SP1、siFXId1-M2SP1、siFXId1-M3SP1、siFXId2-M1SP1、siFXId2-M2SP1、siFXId2-M3SP1、siFXIe1-M1P1、siFXIe1-M2P1、siFXIe1-M3P1、siFXIe2-M1P1、siFXIe2-M2P1、siFXIe2-M3P1、siFXIe1-M1SP1、siFXIe1-M2SP1、siFXIe1-M3SP1、siFXIe2-M1SP1、siFXIe2-M2SP1、siFXIe2-M3SP1、siFXIf1-M1P1、siFXIf1-M2P1、siFXIf1-M3P1、siFXIf2-M1P1、siFXIf2-M2P1、siFXIf2-M3P1、siFXIf1-M1SP1、siFXIf1-M2SP1、siFXIf1-M3SP1、siFXIf2-M1SP1、siFXIf2-M2SP1、siFXIf2-M3SP1、siFXIg1-M1P1、siFXIg1-M2P1、siFXIg1-M3P1、siFXIg2-M1P1、siFXIg2-M2P1、siFXIg2-M3P1、siFXIg1-M1SP1、siFXIg1-M2SP1、siFXIg1-M3SP1、siFXIg2-M1SP1、siFXIg2-M2SP1、siFXIg2-M3SP1、siFXIh1-M1P1、siFXIh1-M2P1、siFXIh1-M3P1、siFXIh2-M1P1、siFXIh2-M2P1、siFXIh2-M3P1、siFXIh1-M1SP1、siFXIh1-M2SP1、siFXIh1-M3SP1、siFXIh2-M1SP1、siFXIh2-M2SP1、siFXIh2-M3SP1、FXIi1-M1P1、siFXIi1-M2P1、siFXIi1-M3P1、siFXIi2-M1P1、siFXIi2-M2P1、siFXIi2-M3P1、siFXIi1-M1SP1、siFXIi1-M2SP1、siFXIi1-M3SP1、siFXIi2-M1SP1、siFXIi2-M2SP1、siFXIi2-M3SP1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，还具有很高的靶mRNA抑制活性。
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
药物组合物
本公开提供了一种药物组合物，所述药物组合物含有如上所述的siRNA作为活性成分和药学上可接受的载体。
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe 3O 4或Fe 2O 3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，可以是各组分常规的含量。在一些实施方案中，siRNA与药学上可接受的载体的重量比可以为1:(1-500)，在一些的实施方案中，上述重量比为1:(1-50)。
在一些实施方案中，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。
在一些实施方案中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺脏或通过喷雾经肺脏给药到其它脏器组织(如肝脏)。在一些实施方案中，所述药物组合物用于静脉注射给药。
在一些实施方案中，所述药物组合物可以为脂质体制剂的形式。在一些实施方案中，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。
在一些实施方案中，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 PCTCN2020091484-appb-000008
其中：
每个X 101或X 102各自独立地是O、S、N-A或C-A，其中A是氢或C 1-C 20烃链；
每个Y 101或Z 101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO 2；
每个R 101、R 102、R 103、R 104、R 105、R 106或R 107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R 102是氢；
并且，如果n或m中的至少一个是2，那么R 103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
Figure PCTCN2020091484-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。
在一些实施方案中，R 103是多胺。在其它实施方案中，R 103是缩酮。在一些实施方案中，在式(201)中的R 101和R 102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方案中，如果n和m中的每一个独立地具有1或3的值，那么R 103可以是下述式(204)-式(213)中的任一个：
Figure PCTCN2020091484-appb-000010
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R 103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
其中，式(201)所示化合物可以根据CN103380113A中的描述制备。
在一些实施方案中，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 PCTCN2020091484-appb-000011
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
所述聚乙二醇化脂质为1,2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方案中，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)。
在一些实施方案中，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。
在一些实施方案中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物中，siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1:1至约1:50、从约1:1至约1:30、从约1:3至约1:20、从约1:4至约1:18、从约1:5至约1:17、从约1:5至约1:15、从约1:5至约1:12、从约1:6至约1:12或从约1:6至约1:10的范围内，例如，本公开的siRNA与全部脂质的重量比为约1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17或1:18。
在一些实施方案中，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。在一些实施方案中，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；在一些实施方案中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200、聚乙二醇300、聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的siRNA溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可 以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1:(2-5)，例如可以为1:4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
siRNA缀合物
本公开提供了一种siRNA缀合物，所述siRNA缀合物含有上述siRNA以及缀合连接至该siRNA的缀合基团。
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。在一些实施方案中，所述靶向基团为1-6个。在一些实施方案中，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。在一些实施方案中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3'末端。
在一些实施方案中，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。在一些实施方案中，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.
在一些实施方案中，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。
在一些实施方案中，所述药学上可接受的靶向基团可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。
在一些实施方案中，所述药学上可接受的靶向基团可以选自以下靶向分子或其衍生物形成的配体中的一种或多种：亲脂分子，例如胆固醇、胆汁酸、维生素(例如维生素E)、不同链长的脂质分子；聚合物，例如聚乙二醇；多肽，例如透膜肽；适配体；抗体；量子点；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；叶酸(folate)；肝实质细胞表达的受体配体，例如去唾液酸糖蛋白、去唾液酸糖残基、脂蛋白(如高密度脂蛋白、低密度脂蛋白等)、胰高血糖素、神经递质(如肾上腺素)、生长因子、转铁蛋白等。
在一些实施方案中，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。在一些实施方案中，至少一个配体是能够与肝细胞表面受体结合的配体。在一些实施方案中，至少一个配体是能够与哺乳动物细胞表面受体结合的配体。在一些实施方案中，至少一个配体是能够与人肝细胞表面受体结合的配体。在一些实施方案中，至少一个配体是能够与肝表面去唾液酸糖蛋白受体(ASGPR)结合的配体。这些配体的种类为本领域技术人员所公知，其作用一般是与靶细胞表面的特异性受体相结合，介导与配体连接的siRNA递送至靶细胞。
在一些实施方案中，所述药学上可接受的靶向基团可以是与哺乳动物肝细胞表面上的去唾液酸糖蛋白受体(ASGPR)结合的任意一种配体。在一些实施方案中，每个配体独立地为去唾液酸糖蛋白，例如去唾液酸血清类粘蛋白(asialoorosomucoid，ASOR)或去唾液酸胎球蛋白(asialofetuin，ASF)。在一些实施方案中，所述配体为糖或糖的衍生物。
在一些实施方案中，至少一个配体是糖。在一些实施方案中，每个配体均是糖。在一些实施方案中，至少一个配体是单糖、多糖、修饰的单糖、修饰的多糖或糖衍生物。在一些实施方案中，至少一个所述配体可以是单糖，双糖或三糖。在一些实施方案中，至少有一个配体是修饰的糖。在一些实施方案中，每一个配体均为修饰的糖。在一些实施方案中，每个配体均独立地选自多糖、修饰的多糖、单糖、修饰的单糖、多糖衍生物或单糖衍生物。在一些实施方案中，每一个或至少一个配体选自于由以下糖所组成的组：葡萄糖及其衍生物、甘露聚糖及其衍生物、半乳糖及其衍生物、木糖及其衍生物、核糖及其衍生物、岩藻糖及其衍生物、乳糖及其衍生物、麦芽糖及其衍生物，阿拉伯糖及其衍生物、果糖及其衍生物和唾液酸。
在一些实施方案中，每个所述配体可独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、 D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳糖胺、N-异丁酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖或L-4-硫代核糖。所述配体的其它选择可参见例如CN105378082A的记载，以引用的方式将其全部公开内容并入本文。
在一些实施方案中，所述siRNA缀合物中药学上可接受的靶向基团可以是半乳糖或N-乙酰半乳糖胺，其中，半乳糖或N-乙酰半乳糖胺分子可以是一价、二价、三价、四价。应当理解的是，这里所述的一价、二价、三价、四价分别指siRNA分子与含有作为靶向基团的半乳糖或N-乙酰半乳糖胺分子的缀合基团形成siRNA缀合物后，该siRNA缀合物中siRNA分子与半乳糖或N-乙酰半乳糖胺分子的摩尔比为1:1、1:2、1:3或1:4。在一些实施方案中，所述药学上可接受的靶向基团是N-乙酰半乳糖胺。在一些实施方案中，当本公开所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价或四价。在一些实施方案中，当本公开所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价。
靶向基团可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些接头、靶向基团的种类以及与siRNA的连接方式，可参见WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。
在一些实施方案中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(301)所示的结构：
Figure PCTCN2020091484-appb-000012
其中，
k为1-3的整数；
L A为具有如式(302)所示结构的包含酰胺键的链状部分，每个所述L A在其两端分别与一个所述靶向基团和所述L C部分通过醚键相连接：
Figure PCTCN2020091484-appb-000013
L B为具有如式(303)所示结构的包含N-酰基吡咯烷的链状部分，所述链状部分在其一端具有羰基并与所述L C部分通过酰胺键相连接，在另一端具有氧基并与所述siRNA通过磷酸酯键相连接：
Figure PCTCN2020091484-appb-000014
L C为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的2-4价连接基团，所述L C经由氧原子与各个所述L A部分通过醚键相连接，并且经由氮原子与所述L B部分通过酰胺键相连接。
在一些实施方案中，当n＝3，L C为基于三羟甲基氨基甲烷的4价连接基团时，由作为接头的-(L A) 3三羟甲基氨基甲烷-L B-连接N-乙酰半乳糖胺分子和siRNA分子所形成的siRNA缀合物，其结构如下式(304)所示：
Figure PCTCN2020091484-appb-000015
式中，双螺旋结构表示siRNA。
同样，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。
在一些实施方案中，本公开所述siRNA的正义链3'末端通过接头-(L A) 3三羟甲基氨基甲烷-L B-与三个N-乙酰半乳糖胺(GalNAc)分子共价缀合，得到siRNA分子与GalNAc分子的摩尔比为1:3的siRNA缀合物，下文也可将其称为(GalNAc) 3-siRNA，其结构如下式(305)所示：
Figure PCTCN2020091484-appb-000016
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3'末端。
在一些实施方案中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(306)所示的结构：
Figure PCTCN2020091484-appb-000017
其中，
l为0-3的整数；
*表示接头上通过醚键与靶向基团连接的位点；
#表示接头上通过磷酸酯键与siRNA连接的位点。
在一些实施方案中，当l＝2时，所述siRNA缀合物具有如式(307)所示的结构：
Figure PCTCN2020091484-appb-000018
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3'末端。
上述缀合物可以通过现有技术中已经详细描述的方法进行合成。例如，WO2015006740A2中详细描述了多种缀合物的制备方法。通过本领域技术人员熟知的方式，获得本公开的siRNA缀合物。如WO2014025805A1中记载了式(305)所示结构的制备方法，Rajeev等人在ChemBioChem2015,16,903-908中描述了式(307)所示结构的制备方法。
在一些实施方案中，所述siRNA缀合物具有如式(308)所示的结构：
Figure PCTCN2020091484-appb-000019
其中：
n1为选自1-3的整数，n3为选自0-4的整数；
每个m1、m2或m3各自独立地为选自2-10的整数；
每个R 10、R 11、R 12、R 13、R 14或R 15各自独立地为H，或选自于由以下基团所组成的组：C 1-C 10烷基、C 1-C 10卤代烷基以及C 1-C 10烷氧基；
R 3为式A59所示结构的基团：
Figure PCTCN2020091484-appb-000020
其中，E 1为OH、SH或BH 2，Nu为本公开的siRNA；
R 2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，R 2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基 苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2，-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
每个L 1是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，L 1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2，-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)。
在一些实施方案中，L 1可选自于由A1-A26基团或其任意连接组合所组成的组，其中A1-A26的结构和定义如下所示：
Figure PCTCN2020091484-appb-000021
Figure PCTCN2020091484-appb-000022
其中，j1为1-20的整数；j2为1-20的整数；
R'为C 1-C 10烷基；
Ra选自式A27-A45基团或其任意连接组合所组成的组：
Figure PCTCN2020091484-appb-000023
Figure PCTCN2020091484-appb-000024
Rb为C 1-C 10烷基；
Figure PCTCN2020091484-appb-000025
表示基团共价连接的位点。
技术人员会理解的是，尽管为了方便起见，L 1被定义为线性亚烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或取代而产生的胺或烯基。为了本公开内容的目的，L 1的长度是连接两个连接点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)计为一个原子。
M 1表示靶向基团，其定义和可选择的范围与上述靶向基团相同。在一些实施方案中，每个M 1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。
当M 1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体时，在一些实施方案中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述缀合物中M 1靶向基团的个数至少为2；在一些实施方案中，n1+n3≥2，这样可以使得M 1靶向基团的个数至少为3，从而使得M 1靶向基团与肝表面去唾液酸糖蛋白受体更容易结合，进而促进所述缀合物通过内吞作用进入细胞。实验表明，当M 1靶向基团的个数大于3个时，M 1靶向基团与肝表面去唾液酸糖蛋白受体结合的容易程度增加并不明显，因此，从合成容易程度、结构/工艺成本和递送效率等多方面综合考虑，在一些实施方案中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。
在一些实施方案中，每m1、m2或m3各自独立地选自2-10的整数时，可以使多个M 1靶向基团之间的空间位置适合M 1靶向基团与肝表面去唾液酸糖蛋白受体的结合，为了使本公开提供的siRNA缀合物更为简单，更容易合成和/或降低成本，在一些实施方案中，每个m1、m2和m3各自独立地为2-5的整数，在一些实施方案中，m1＝m2＝m3。
本领域技术人员可以理解，当每个R 10、R 11、R 12、R 13、R 14或R 15各自独立地选自H、C 1-C 10烷基、C 1-C 10卤代烷基、以及C 1-C 10烷氧基中的一种时，不会改变本公开的siRNA缀合物的性质，均可以实现本公开的目的。在一些实施方案中，每个R 10、R 11、R 12、R 13、R 14或R 15各自独立地选自H、甲基或乙基。在一些实施方案中，每个R 10、R 11、R 12、R 13、R 14和R 15均为H。
R 3为式A59所示结构的基团，其中，E 1为OH、SH或BH 2，基于制备原料易获取性的考虑，在一些实施方案中，E 1为OH或SH。
R 2的选择是为了实现与含氮骨架上的N原子与A59的连接。在本公开的上下文中，“含氮骨架”是指连接有R 10、R 11、R 12、R 13、R 14和R 15的碳原子与N原子互相连接的链状结构。因此，R 2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N原子的连接基团。在一些实施方案中，在通过固相合成的工艺制备式(308)所示的siRNA缀合物的情况下，R 2基团中需要同 时含有与含氮骨架上的N原子连接的连接位点和与R 3中的P原子相连接的连接位点。在一些实施方案中，R 2中所述与含氮骨架上的N原子连接的位点与N原子形成酰胺键，所述与R 3上的P原子连接的位点与P原子形成磷酸酯键；在一些实施方案中，R 2可以是B5、B6、B5'或B6'：
Figure PCTCN2020091484-appb-000026
其中，
Figure PCTCN2020091484-appb-000027
表示基团共价连接的位点。
q 2的取值范围可以是1-10的整数，在一些实施方案中，q 2为1-5的整数。
L 1的作用是将M 1靶向基团与含氮骨架上的N原子连接，为式(308)所示的siRNA缀合物提供肝靶向功能。在一些实施方案中，L 1选自式A1-A26基团中的一种或多种的连接组合。在一些实施方案中，L 1选自A1、A4、A5、A6、A8、A10、A11和A13中的一种或多种的连接组合。在一些实施方案中，L 1选自A1、A4、A8、A10和A11中至少2个的连接组合。在一些实施方案中，L 1选自A1、A8、A10中至少2个的连接组合。
在一些实施方案中，L 1的长度可以为3-25个原子，3-20个原子、4-15个原子或5-12个原子。在一些实施方案中，L 1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。
在一些实施方案中，j1为2-10的整数，在一些实施方案中，j1为3-5的整数。在一些实施方案中，j2为2-10的整数，在一些实施方案中，j2为3-5的整数。R'为C 1-C 4烷基，在一些实施方案中，R'为甲基、乙基和异丙基中的一种。Ra为A27、A28、A29、A30和A31中的一种，在一些实施方案中，Ra为A27或A28。Rb为C 1-C 5烷基，在一些实施方案中，Rb为甲基、乙基、异丙基和丁基中的一种。在一些实施方案中，在式A1-A26中各自对j1、j2、R'、Ra、Rb进行选择，以实现M 1靶向基团与含氮骨架上的N原子连接，并使M 1靶向基团之间的空间位置更适合M 1靶向基团与肝表面去唾液酸糖蛋白受体结合。
在一些实施方案中，该缀合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：
Figure PCTCN2020091484-appb-000028
Figure PCTCN2020091484-appb-000029
Figure PCTCN2020091484-appb-000030
Figure PCTCN2020091484-appb-000031
Figure PCTCN2020091484-appb-000032
Figure PCTCN2020091484-appb-000033
在一些实施方案中，式A59中的P原子可以连接到siRNA序列中任何可能的位置，例如，式A59中的P原子可以连接到siRNA正义链或反义链的任何一个核苷酸上；在一些实施方案中，式A59中的P原子连接到siRNA正义链的任何一个核苷酸上。在一些实施方案中，式A59中的P原子连接到siRNA正义链或反义链的端部；在一些实施方案中，式A59中的P原子连接到siRNA正义链的端部。所述端部指所述正义链或所述反义链中从其一端起算的前4个核苷酸。在一些实施方案中，式A59中的P原子连接到siRNA正义链或反义链的末端；在一些实施方案中，式A59中的P原子连接到siRNA正义链的3'末端。在连接至siRNA的正义链的上述位置的情况下，式 (308)所示的siRNA缀合物进入细胞后，在解旋时，可以释放出单独的siRNA反义链，以阻断FXI mRNA翻译蛋白质的过程，抑制FXI基因表达。
在一些实施方案中，式A59中的P原子可以连接到siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5'位、核苷酸的2'位、核苷酸的3'位或核苷酸的碱基上。在一些实施方案中，式A59中的P原子可通过形成磷酸二酯键连接至所述siRNA中的核苷酸的2'位、3'位或5'位。在一些实施方案中，式A59中的P原子连接在siRNA正义链3'末端核苷酸的3'羟基脱氢后形成的氧原子上(此时，A59中的P原子也可以看作是siRNA中含有的磷酸基团中的P原子)，或者式A59中的P原子通过取代siRNA正义链中的一个核苷酸的2'-羟基中的氢与核苷酸连接，或者式A59中的P原子通过取代siRNA正义链5'末端核苷酸的5'羟基中的氢与核苷酸连接。
本公开的发明人意外发现，本公开的siRNA缀合物在具有显著提高的血浆中稳定性、低脱靶效应的同时，还表现出较高的FXI mRNA沉默活性。在一些实施方案中，本公开的siRNA可以为表1a-1i中示出的siRNA中的一种。含有这些siRNA的siRNA缀合物表现出更高的FXI mRNA沉默活性。
表1a：本公开的第一种siRNA序列
Figure PCTCN2020091484-appb-000034
Figure PCTCN2020091484-appb-000035
表1b：本公开的第二种siRNA序列
Figure PCTCN2020091484-appb-000036
Figure PCTCN2020091484-appb-000037
表1c：本公开的第三种siRNA序列
Figure PCTCN2020091484-appb-000038
Figure PCTCN2020091484-appb-000039
Figure PCTCN2020091484-appb-000040
表1d：本公开的第四种siRNA序列
Figure PCTCN2020091484-appb-000041
Figure PCTCN2020091484-appb-000042
表1e：本公开的第五种siRNA序列
Figure PCTCN2020091484-appb-000043
Figure PCTCN2020091484-appb-000044
表1f：本公开的第六种siRNA序列
Figure PCTCN2020091484-appb-000045
Figure PCTCN2020091484-appb-000046
Figure PCTCN2020091484-appb-000047
表1g：本公开的第七种siRNA序列
Figure PCTCN2020091484-appb-000048
Figure PCTCN2020091484-appb-000049
表1h：本公开的第八种siRNA序列
Figure PCTCN2020091484-appb-000050
Figure PCTCN2020091484-appb-000051
表1i：本公开的第九种siRNA序列
Figure PCTCN2020091484-appb-000052
Figure PCTCN2020091484-appb-000053
Figure PCTCN2020091484-appb-000054
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接；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。在一些实施方案中，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修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。
本公开所述siRNA或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子、铵离子NH 4 +、有机铵阳离子中的一种。出于提高溶解性考虑，在一种实施方案中，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。
式(308)所示的siRNA缀合物的制备
可以采用任意合理的合成路线制备式(308)所示的siRNA缀合物。
在一些实施方案中，式(308)所示的siRNA缀合物可以采用如下方法制备，该方法包括在亚磷酰胺固相合成的条件下，分别按照siRNA正义链和反义链的核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；分离出siRNA的正义链和反义链，退火，其中，所述siRNA为上述本公开的siRNA；
并且，该方法还包括在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与核苷单体或连接在固相载体上的核苷酸序列接触，使式(321)所示的化合物经偶联反应连接至核苷酸序列。下文中，式(321)所示的化合物也称作缀合分子。
Figure PCTCN2020091484-appb-000055
其中：
R 4为能够结合至式(308)所示的化合物中Nu代表的siRNA的基团。在一些实施方案中，R 4为能够通过共价键结合至Nu代表的siRNA的基团。在一些实施方案中，R 4为能够经反应而通过磷酸二酯键缀合至Nu代表的siRNA的任意官能团的基团；
每个S 1独立地是M 1中全部活性羟基被YCOO-基团取代而形成的基团，其中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；在一些实施方案中，Y为甲基。
n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15、L 1、M 1各自的定义和可选择的范围如前所述。
R 4的选择是为了实现与含氮骨架上的N原子的连接，并且为合成式(308)所示的siRNA缀合物提供合适的反应位点。在一些实施方案中，R 4中包括R 2连接基团或经保护的R 2连接基团，以及可通过反应与siRNA形成A59所示结构的官能团。
在一些实施方案中，R 4包含可与Nu代表的siRNA或核苷单体上的基团形成亚磷酸酯的第1官能团以及可与羟基或氨基反应形成共价键的第2官能团或者含有由所述共价键连接的固相载体。在一些实施方案中，所述第1官能团为亚磷酰胺、羟基或被保护的羟基。在一些实施方案中， 所述第2官能团为亚磷酰胺、羧基或羧酸盐。在一些实施方案中，所述第2官能团为经由共价键连接至分子其他部分的固相载体，所述共价键由羟基或氨基形成。在一些实施方案中，所述固相载体经由磷酸酯键、羧酸酯键或酰胺键连接。在一些实施方案中，所述固相载体为树脂。
在一些实施方案中，所述第1官能团含有羟基、-OR k或式(C3)所示的基团；所述第2官能团含有式(C1)、(C2)、(C3)、(C1')或(C3')所示的结构：
Figure PCTCN2020091484-appb-000056
式中，q 1为1-4的整数，X为O或NH，M +为阳离子，R k为羟基保护基团，SPS表示固相载体，
Figure PCTCN2020091484-appb-000057
表示基团连接共价部分的位点。
在一些实施方案中，所述第1官能团含有亚磷酰胺基团，如式(C3)所示，该亚磷酰胺基团可以与核苷酸上的任意位置的羟基，如2'位羟基或3'位羟基发生偶联反应形成亚磷酸酯，并经氧化或硫化形成式A59所示的磷酸二酯键或硫代磷酸酯键，将缀合分子缀合至siRNA。此时，即使所述第2官能团并不存在，式(321)所示化合物也能够缀合至核苷酸，不影响式(308)所示的siRNA缀合物的获得。在此情况下，在经由亚磷酰胺固相合成等方法获得siRNA的正义链或反义链后，使式(321)所示化合物与核苷酸序列中末端核苷酸上的羟基反应，并在后续的氧化或硫化过程中形成磷酸二酯键连接或硫代磷酸酯连接，将式(321)所示化合物缀合至siRNA。
在一些实施方案中，所述第1官能团含有被保护的羟基。在一些实施方案中，所述第2官能团包含可与固相载体反应的基团，所述反应提供包含固相载体的缀合分子。在一些实施方案中，所述第2官能团含有羧基、羧酸盐或亚磷酰胺，如式(C1)、(C2)或(C3)所示，当所述第2官能团包含羧基或羧酸盐时，式(321)所示化合物与固相载体，例如树脂上的羟基或氨基进行酯化反应或酰胺化反应，形成经羧酸酯键连接的包含固相载体的缀合分子。当所述第2官能团包含亚磷酰胺官能团时，式(321)所示化合物与通用固相载体，例如树脂上的羟基发生偶联反应，并经氧化形成经磷酸二酯键连接的包含固相载体的缀合分子。随后，以上述连接固相载体后的产物作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。在亚磷酰胺固相合成过程中，所述第1官能团发生脱保护，随后在偶联反应条件下与核苷单体上的亚磷酰胺基团发生偶联。
在一些实施方案中，所述第1官能团含有羟基或被保护的羟基；所述第2官能团含有经羧酸酯键连接的固相载体或经酰胺键连接的固相载体或者经磷酸酯键连接的固相载体，如式(C1')或(C3')所示。此时，由式(321)所示化合物代替固相载体作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。
在一些实施方案中，羧酸盐可以表示为-COO -M +，其中，M +是阳离子，例如选自金属阳离子、铵阳离子NH 4 +、有机铵阳离子中的一种。在一种实施方案中，所述金属离子选自碱金属离子中的一种，如K +或Na +。出于提高溶解性、使反应顺利进行的考虑，在一些实施方案中，有机铵离子为三级胺形成的铵阳离子或季铵阳离子，如，三乙胺形成的铵离子或N,N-二异丙基乙胺形成的铵离子。在一些实施方案中，羧酸盐是三乙胺羧酸盐或N,N-二异丙基乙胺羧酸盐。
在一些实施方案中，R 4含有式(B9)、(B10)、(B9')、(B10')、(B11)、(B12)、(B11')或(B12')所示的结构：
Figure PCTCN2020091484-appb-000058
其中，q 1为1-4的整数，q 2为1-10的整数，X为O或NH，M +为阳离子，R k为羟基保护基团，SPS表示固相载体，
Figure PCTCN2020091484-appb-000059
表示基团连接共价部分的位点。在一些实施方案中，q 1为1或2。在一些实施方案中，q 2为1-5的整数。在一些实施方案中，R 4含有式(B9)或(B10)所示的结构。在一些实施方案中，R 4含有式(B11)或(B12)所示的结构。
在一些实施方案中，R k是Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4'-双甲氧基三苯甲基)、TMTr(4,4',4”-三甲氧基三苯甲基)中的一种或多种。在一些实施方案中，R k可以是DMTr，即4,4'-双甲氧基三苯甲基(4,4'-dimethoxytrityl)。
L 1的定义如前所述。
在一些实施方案中，L 1被用于将M 1靶向基团连接至含氮骨架上的N原子，从而为式(308)所示的siRNA缀合物提供肝靶向功能。在一些实施方案中，L 1包含A1-A26中的任一个或其组合。
根据上述描述，本领域技术人员容易理解的是，相较于本领域公知的亚磷酰胺固相合成方法而言，可通过上述第1官能团以及任选的第2官能团，获得将缀合分子连接至核苷酸序列的任意可能的位置的式(308)所示的siRNA缀合物，例如，缀合分子连接至核苷酸序列的端部，缀合分子连接至核苷酸序列的末端。相应地，除非另有说明，以下涉及缀合物和/或缀合分子的制备的描述中，当提及“脱保护”、“偶联”、“盖帽”、“氧化”、“硫化”等反应时，应当理解为本领域公知 的亚磷酰胺核酸固相合成方法中所涉及的反应条件和试剂也同样适用于这些反应。示例性的反应条件和试剂将在后文详细描述。
在一些实施方案中，每个S 1独立地是M 1。在一些实施方案中，每个S 1独立地是M 1中至少一个活性羟基被羟基保护基团保护而形成的基团。在一些实施方案中，每个S 1独立地是M 1中任何存在的活性羟基全部被羟基保护基团保护而形成的基团。在一些实施方案中，任何本领域技术人员已知的羟基保护基团均可被用于保护M 1中的活性羟基。在一些实施方案中，被保护的羟基可以式YCOO-表示，其中，每个Y独立地选自于由C 1-C 10烷基和C 6-C 10芳基所组成的组，所述C 1-C 10烷基和C 6-C 10芳基任选地被一个或多个取代基取代，所述取代基选自于由卤素和C 1-C6烷基所组成的组。在一些实施方案中，每个Y独立地选自于由以下基团所组成的组：甲基、三氟甲基、二氟甲基、单氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤苯基，以及C 1-C 6烷基苯基。
在一些实施方案中，每个S 1各自独立地选自于由式A46-A54所组成的组：
Figure PCTCN2020091484-appb-000060
在一些实施方案中，S 1为式A49或A50。
在一些实施方案中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；在一些实施方案中，Y为甲基。
如前所述，式(308)所示的siRNA缀合物的制备方法还包括以下步骤：合成siRNA的另一链(例如，当上述步骤合成了连接有缀合分子的siRNA正义链时，还包括按照固相合成方法合成siRNA的反义链，反之亦然)，分离正义链和反义链，以及退火。具体地，在分离步骤中，连接至核苷酸序列和/或缀合分子的固相载体被切割下来，同时必要的保护基团被脱除(此时，式(321)所示化合物中的各S 1基团转化为对应的M 1靶向基团)，获得连接有缀合分子的siRNA正义链(或反义链)以及对应的反义链(或正义链)，正义链与反义链退火形成双链RNA结构，获得式(308)所示的siRNA缀合物。
在一些实施方案中，式(308)所示的siRNA缀合物的制备方法包含以下步骤：在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与正义链或反义链的3'端的第一个核苷单体 接触，使式(321)所示的化合物连接上序列中第一个核苷酸，在亚磷酰胺固相合成的条件下，按照期望的正义链或反义链核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，合成siRNA的正义链或反义链；其中，式(321)所示化合物为R 4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基，第2官能团具有如式(C1')或(C3')所示结构的化合物，与第一个核苷单体连接前，式(321)所示化合物经过脱保护；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；得到连接有缀合基团的核酸的正义链或反义链；在亚磷酰胺固相合成的条件下，按照反义链或正义链核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，合成核酸的反义链或正义链；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；脱除保护基并与固相载体切割，分离纯化获得正义链和反义链，退火。
在一些实施方案中，式(308)所示的siRNA缀合物的制备方法包含以下步骤：按照该双链siRNA中正义链或反义链的核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，合成正义链和反义链，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应，得到连接在固相载体上的正义链和连接在固相载体上的反义链；在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与连接在固相载体上的正义链或连接在固相载体上的反义链接触，将式(321)所示化合物连接至正义链或反义链，其中，式(321)所示化合物是R 4中含有第1官能团，第1官能团为亚磷酰胺基团的式(321)所示化合物；脱除保护基并与固相载体切割，分别分离纯化，获得siRNA的正义链或反义链，退火，其中，所述siRNA的正义链或反义链上连接有缀合基团。
在一些实施方案中，式A59中的P原子连接至siRNA中的正义链的3'末端，式(308)所示的siRNA缀合物的制备方法包括：
(1)脱除式(321)所示化合物(其中，式(321)所示化合物为R 4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基OR k，第2官能团具有如式(C1')或(C3')所示结构的化合物)中的羟基保护基团R k；在偶联反应条件和偶联试剂存在下，将脱保护得到的产物与核苷单体接触，得到通过缀合分子连接至固相载体的核苷单体；
(2)以该通过缀合分子连接至固相载体的核苷单体起始，按照3'-5'的方向通过亚磷酰胺固相合成方法合成siRNA的正义链；
(3)通过亚磷酰胺固相合成方法，合成siRNA的反义链；
(4)分离出siRNA的正义链和反义链并退火，获得式(308)所示的siRNA缀合物。
其中，在步骤(1)中，脱除上述式(321)所示化合物中的保护基团R k的方法包括在脱保护条件下，将式(321)所示化合物与脱保护试剂接触。脱保护条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为30-300秒，在一些实施方案中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方案中为二氯乙酸。脱保护试剂与式(321)所示化合物的摩尔比为10:1-1000:1，在一些实施方案中为50:1-500:1。
所述偶联反应条件和偶联试剂可使用任何适合于上述偶联反应的条件和试剂。在一些实施方案中，可使用与所采用的固相合成方法中的偶联反应相同的条件与试剂。
在一些实施方案中，所述偶联反应的条件包括反应温度为0-50℃，在一些实施方案中为15-35℃。式(321)所示化合物与核苷单体的摩尔比为1:1-1:50，在一些实施方案中为1:2-1:5；式(321)所示化合物和偶联试剂的摩尔比可以为1:1-1:50，在一些实施方案中为1:3-1:10，反应时间为200-3000秒，在一些实施方案中为500-1500秒。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，在一些实施方案中为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，在一些实施方案中为无水乙腈。相对于式(321)所示化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方案中为5-20L/mol。
在步骤(2)中，通过亚磷酰胺核酸固相合成的方法，利用上述步骤制备的通过缀合分子连接至固相载体的核苷单体起始，按照3'-5'的方向合成第二种siRNA缀合物的正义链SS。此时，缀合基团连接至所得到的正义链的3'末端。
步骤(2)和(3)中所述固相合成的其它条件，包括核苷单体脱保护条件、脱保护试剂种类和用量、偶联反应条件、偶联试剂的种类和用量、盖帽反应的条件、盖帽试剂的种类和用量、氧化反应条件、氧化试剂种类和用量、硫化反应条件、硫化试剂种类和用量采用本领域中常规使用的各种试剂、用量和条件。
例如，在一些实施方案中，步骤(2)和(3)中所述固相合成可使用如下条件：
核苷单体脱保护条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为30-300秒，在一些实施方案中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸、中的一种或多种，在一些实施方案中为二氯乙酸。脱保护试剂与固相载体上4,4'-二甲氧基三苯甲基保护基的的摩尔比可以为2:1-100:1，在一些实施方案中为3:1-50:1。
偶联反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，固相载体上连接的核酸序 列与核苷单体的摩尔比可以为1:1-1:50，在一些实施方案中为1:5-1:15；固相载体上连接的核酸序列和偶联试剂的摩尔比为1:1-1:100，在一些实施方案中为1:50-1:80，反应时间和偶联试剂的选择与前述相同。
盖帽反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为5-500秒，在一些实施方案中为10-100秒，盖帽试剂的选择与前述相同。盖帽试剂的总量与固相载体上连接的核酸序列的摩尔比为1:100-100:1，在一些实施方案中为1:10-10:1。在盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑的情况下，乙酸酐、N-甲基咪唑以及固相载体上连接的核酸序列的摩尔比可为1:1:10-10:10:1，在一些实施方案中为1:1:2-2:2:1。
氧化反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为1-100秒，在一些实施方案中为5-50秒，氧化试剂在一些实施方案中为碘(在一些实施方案中，以碘水的形式提供)。氧化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比可以为1:1-100:1，在一些实施方案中为5:1-50:1。在一些实施方案中，所述氧化反应在四氢呋喃:水:吡啶＝3:1:1-1:1:3的混合溶剂中进行。硫化反应条件包括温度为0-50℃，在一些实施方案中为15-35℃，反应时间为50-2000秒，在一些实施方案中为100-1000秒，硫化试剂在一些实施方案中为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比为10:1-1000:1，在一些实施方案中为10:1-500:1。在一些实施方案中，所述硫化反应在乙腈:吡啶＝1:3-3:1的混合溶剂中进行。
在将所有核苷单体连接之后，退火之前，该方法还包括分离出siRNA的正义链和反义链。分离的方法为本领域技术人员所公知，一般包括将合成得到的核苷酸序列从固相载体上切割下来，脱除碱基上、磷酸基上和配体上的保护基团，纯化和脱盐。
将合成得到的核苷酸序列从固相载体上切割下来，并脱除碱基上、磷酸基上和配体上的保护基团可按照siRNA合成中常规的切割和脱保护方法进行。例如，将得到的连接有固相载体的核苷酸序列与浓氨水接触；在脱保护的过程中，A46-A54基团的保护基团YCOO-转化为羟基，S 1基团转化为相应的M 1基团，生成式(308)所示的siRNA缀合物。其中，所述浓氨水可以是25-30重量％的氨水，浓氨水的用量与目标siRNA序列相比可以为0.2ml/μmol-0.8ml/μmol。
在所合成的核苷酸序列上存在至少一个2'-TBDMS保护时，所述方法还包括将脱除了固相载体的核苷酸序列与三乙胺三氢氟酸盐接触，以脱除该2'-TBDMS保护。此时，所得到的目标siRNA序列中的相应核苷酸具有游离的2'-羟基。三乙胺三氢氟酸盐纯品的用量与目标siRNA序列相比可以为0.4ml/μmol-1.0ml/μmol。这样即可得到式(308)所示的siRNA缀合物。
纯化和脱盐的方法是本领域技术人员熟知的。例如，可利用制备型离子色谱纯化柱，通过NaBr或NaCl的梯度洗脱，完成核酸的纯化；产品收集合并后，可采用反相色谱纯化柱进行脱盐。
这样得到的式(308)所示的siRNA缀合物中，核苷酸之间的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基本与钠离子结合，式(308)所示的siRNA缀合物基本以钠盐形式存在。可以采用熟知的离子交换方法，用氢离子和/或其他阳离子取代所述钠离子，得到其他形式的式(308)所示的siRNA缀合物。所述阳离子如前所述。
在合成过程中，可随时对核酸序列的纯度和分子量进行检测，更好地把控合成质量，此类检测的方法为本领域技术人员所公知。例如，可通过离子交换色谱检测核酸纯度，并通过液质联用色谱(LC-MS)测定分子量。
退火的方法也是本领域技术人员熟知的。例如，可简单地将所合成的正义链(S链)与反义链(AS链)以等摩尔比混合在注射用水中加热至70-95℃，随后室温冷却，使其通过氢键形成双链结构。这样即可得到式(308)所示的siRNA缀合物。
在获得所述缀合物后，在一些实施方案中，还可利用例如液质联用色谱等方法，通过分子量检测等方式对所合成的式(308)所示的siRNA缀合物进行表征，确定所合成的siRNA缀合物为目标设计的式(308)所示的siRNA缀合物，且所合成的siRNA的序列为期望的siRNA的序列，例如为表1中所列的序列之一。
式(321)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在酯化反应条件下，以及在碱和酯化催化剂存在下，将式(313)所示化合物与环状酸酐接触，离子交换，分离得到式(321)所示化合物：
Figure PCTCN2020091484-appb-000061
其中，n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15、L 1、S 1各自的定义和可选择的范围如前所述；
R 6为提供式(321)中R 4的基团；在一些实施方案中，R 6具有式(A61)所示的结构：
Figure PCTCN2020091484-appb-000062
其中，R i为能够实现与含氮骨架上的N原子连接、与R kO连接并且连接有一个游离羟基的任意基团，R k为羟基保护基团。此时，所获得的是R 4中含有作为羟基保护基团的第1官能团和第2官能团，所述第2官能团含有如式(C1)或(C2)所示结构的式(321)所示化合物。
所述酯化反应条件包括反应温度为0-100℃，反应时间为8-48小时，在一些实施方案中，所述酯化反应条件为反应温度为10-40℃，反应时间为20-30小时。
在一些实施方案中，所述有机溶剂包含环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。在一些实施方案中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种。在一些实施方案中，所述有机溶剂为二氯甲烷。相对于所述式(313)所示化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方案中为5-20L/mol。
在一些实施方案中，所述环状酸酐为丁二酸酐、戊二酸酐、己二酸酐或庚二酸酐中的一种，在一些实施方案中为丁二酸酐。所述环状酸酐与所述式(313)所示化合物的摩尔比为1:1-10:1，在一些实施方案中为2:1-5:1。
所述酯化催化剂可以是任何对该酯化反应起到催化作用的催化剂，例如该催化剂可以是4-二甲氨基吡啶。所述催化剂与式(313)所示化合物的摩尔比为1:1-10:1，在一些实施方案中为2:1-5:1。
在一些实施方案中，所述碱可以是任意的无机碱，有机碱或者它们的结合。考虑溶解性和产物稳定性，所述碱可以是例如三级胺。在一些实施方案中，所述三级胺为三乙胺或N,N-二异丙基乙胺。所述三级胺与式(313)所示化合物的摩尔比为1:1-20:1，在一些实施方案中为3:1-10:1。
所述离子交换作用是将式(321)所示化合物转化为期望的羧酸或羧酸盐的形式，离子交换的方法为本领域技术人员所公知，可以使用合适的离子交换溶液和交换条件，得到具有M +阳离子的缀合分子，在此不做详述。在一些实施方案中，所述离子交换反应使用三乙胺磷酸盐溶液进行，所述三乙胺磷酸盐溶液的浓度为0.2-0.8M，在一些实施方案中，所述三乙胺磷酸盐溶液的浓度为0.4-0.6M，相对于式(313)所示化合物，所述三乙胺磷酸盐溶液的用量为3-6L/mol，在进一步的实施方案中为4-5L/mol。
可使用任何合适的分离方法从反应混合物中分离式(321)所示化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(321)所示化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用含1wt‰三乙胺的二氯甲烷:甲醇＝100:18-100:20梯度洗脱；或者(2)反相纯化：C18、C8反相填料，使用甲醇:乙腈＝0.1:1-1:0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(321)所示化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方案中，式(321)所示化合物的制备方法还进一步包括在缩合反应条件下，在有机溶剂中，在缩合剂、缩合催化剂和三级胺的存在下，将上述离子交换反应得到的产物进一步与含有氨基或羟基的固相载体进行接触。此时，所获得的是R 4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C1')所示结构的式(321)所示化合物。
所述固相载体为固相合成siRNA中所用的载体中的一种，其中的一些为本领域技术人员所公知。例如,所述固相载体可以选自含有活性羟基或氨基官能团的固相载体，在一些实施方案中，所述固相载体为氨基树脂或羟基树脂。在一些实施方案中，所述氨基或羟基树脂具有如下参数：粒径100-400目(mesh)，表面氨基或羟基载量为0.2-0.5mmol/g。所述式(321)所示化合物与固相载体的用量比为10-400μmol化合物/每克固相载体(μmol/g)。在一些实施方案中，所述式(321)所示化合物与固相载体的用量比为50-200μmol/g。
所述有机溶剂可以是本领域技术人员已知的任何合适的溶剂或混合溶剂。在一些实施方案中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。在一些实施方案中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种。在一些实施方案中，所述有机溶剂为乙腈。相对于式(321)所示化合物，所述有机溶剂的用量为20-200L/mol，在一些实施方案中为50-100L/mol。
在一些实施方案中，所述缩合剂可以是苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸酯/盐(benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate，PyBop)、3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮(3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one，DEPBT)和/或O-苯并三唑-四甲基脲六氟磷酸酯/盐(O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate)，在一些实施方案中，所述缩合剂为O-苯并三氮唑-四甲基脲六氟磷酸盐/酯。所述缩合剂与式(321)所示化合物的摩尔比为1:1-20:1，在其它实施方案中为1:1-5:1。
在一些实施方案中，所述三级胺为三乙胺和/或N,N-二异丙基乙胺，在一些实施方案中为N,N-二异丙基乙胺；所述三级胺与式(321)所示化合物的摩尔比为1:1-20:1，在一些实施方案中为1:1-5:1。
在一些实施方案中，式(321)所示化合物的制备方法还可以包括在盖帽反应条件下，在有机溶剂中，将得到的缩合产物与盖帽试剂和酰化催化剂接触，分离得到式(321)所示化合物。所述盖帽反应的作用在于除去任何尚未反应完全的活性反应官能团，以避免在后续反应中产生不必要的副产物。所述盖帽反应的条件包括反应温度为0-50℃，在一些实施方案中为15-35℃，反应的时间为1-10h，在一些实施方案中为3-6h。盖帽试剂可以使用siRNA固相合成中所使用的盖帽试剂，siRNA固相合成中所使用的盖帽试剂为本领域技术人员所公知。
在一些实施方案中，所述盖帽试剂由盖帽试剂1(cap1)和盖帽试剂2(cap2)组成，其中，盖帽试剂1为N-甲基咪唑，在一些实施方案中以N-甲基咪唑的吡啶/乙腈混合溶液形式提供，其中，吡啶与乙腈的体积比为1:10-1:1，在一些实施方案中为1:3-1:1，吡啶与乙腈的总体积与N-甲基咪唑的体积比为1:1-10:1，在一些实施方案中为3:1-7:1。所述盖帽试剂2为乙酸酐。在一些实施方案中，所述盖帽试剂2以乙酸酐的乙腈溶液形式提供，其中，乙酸酐和乙腈的体积比为1:1-1:10，在进一步的实施方案中为1:2-1:6。
在一些实施方案中，所述N-甲基咪唑的吡啶/乙腈混合溶液的体积与式(321)所示化合物的质量之比为5ml/g-50ml/g，在一些实施方案中为15ml/g-30ml/g。所述乙酸酐的乙腈溶液的体积与式(321)所示化合物的质量之比为0.5ml/g-10ml/g，在一些实施方案中为1ml/g-5ml/g。
在一些实施方案中，盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑。在一些实施方案中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。在一些实施方案中，所述有机溶剂为乙腈。相对于式(321)所示化合物，所述有机溶剂的用量为10-50L/mol，在一些实施方案中为5-30L/mol。
在一些实施方案中，所述酰化催化剂可以选自任何可用于酯化缩合或酰胺化缩合的催化剂，例如碱性杂环化合物。在一些实施方案中，所述酰化催化剂为4-二甲氨基吡啶。所述催化剂与式(321)所示化合物的质量之比为0.001:1-1:1，在一些实施方案中为0.01:1-0.1:1。
在一些实施方案中，可使用任何合适的分离方法从反应混合物中分离式(321)所示化合物。在一些实施方案中，可通过以有机溶剂充分洗涤，并过滤，去除未反应的反应物、过量的盖帽试剂及其它杂质，得到式(321)所示化合物，所述有机溶剂选自乙腈、二氯甲烷、甲醇，在一些实施方案中为乙腈。
在一些实施方案中，式(321)所示缀合分子的制备方法包括在有机溶剂中，在偶联反应条件下，以及在偶联试剂存在下，将式(313)所示化合物与亚磷酰二胺接触，分离得到式(321)所示化合物。此时，所获得的是R 4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C3)所示结构的式(321)所示化合物。
在一些实施方案中，偶联反应条件包括温度可以为0-50℃，例如为15-35℃，式(313)所示化合物与亚磷酰二胺的摩尔比可以为1:1-1:50，例如为1:5-1:15；式(313)所示化合物和偶联试剂的摩尔比可以为1:1-1:100，例如为1:50-1:80；反应时间可以为200-3000秒，例如为500-1500秒。所述亚磷酰二胺例如可使用双(二异丙基氨基)(2-氰基乙氧基)膦，其可商购获得或按照本领域中公知的方法合成获得。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，例如为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，例如为无水乙腈。在一些实施方案中，相对于式(313)所示化合物，所述有机溶剂的用量为3-50L/mol，例如可以为5-20L/mol。通过进行该偶联反应，式(313)所示化合物中的羟基与亚磷酰二胺反应形成亚磷酰胺基团。在一些实施方案中，可以直接除去溶剂得到式(321)所示化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方案中，式(321)所示化合物的制备方法还进一步包括以下步骤：在偶联反应条件下，在有机溶剂中，以及在偶联试剂存在下，将分离得到的产物进一步与含有羟基的固相载体进行接触。随后，经盖帽反应、氧化反应，分离得到式(321)所示化合物。此时，所获得的是R 4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团具有如式(C3')所示结构的式(321)所示化合物。
在一些实施方案中，所述固相载体为本领域中公知的可用于核酸固相合成的固相载体，例如， 可以是经脱保护反应后的市售的通用固相载体(
Figure PCTCN2020091484-appb-000063
HL UnyLinker TM 300 Oligonucleotide Synthesis Support，Kinovate Life Sciences公司，结构如式B80所示)：
Figure PCTCN2020091484-appb-000064
脱保护反应为本领域技术人员所公知。在一些实施方案中，脱保护条件包括温度为0-50℃，例如为15-35℃；反应时间为30-300秒，例如为50-150秒。脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方案中，脱保护试剂为二氯乙酸。脱保护试剂与固定相上的-DMTr(4,4'-二甲氧基三苯甲基)保护基的摩尔比为2:1-100:1，例如为3:1-50:1。通过进行所述脱保护，在所述固相载体表面上获得具有反应活性的游离羟基，便于进行后续的偶联反应。
偶联反应条件以及偶联试剂的选择可如上所述。通过进行该偶联反应，脱保护反应中形成的游离羟基与亚磷酰胺基团反应形成亚磷酸酯连接。
在一些实施方案中，盖帽反应条件包括温度为0-50℃，例如为15-35℃，反应时间为5-500秒，例如为10-100秒，所述盖帽反应在盖帽试剂存在下进行。盖帽试剂的选择和用量可如上所述。
氧化反应条件包括温度为0-50℃，例如可以为15-35℃，反应时间为1-100秒，例如可以为5-50秒，氧化试剂例如可以为碘(在一些实施方案中，以碘水的形式提供)。在一些实施方案中，氧化试剂与连接至固相载体的核酸序列的摩尔比为1:1-100:1，例如可以为5:1-50:1。在一些实施方案中，所述氧化反应在四氢呋喃:水:吡啶＝3:1:1-1:1:3的混合溶剂中进行。
在一些实施方案中，R 6为式B7或B8基团中的一种，
Figure PCTCN2020091484-appb-000065
其中q 2的定义如前所述，
此时，式(313)所示化合物可以通过以下制备方法得到：在有机溶剂中，在酰胺化反应条件下，以及在酰胺化反应缩合剂和三级胺存在下，将式(314)所示化合物与式(A-1)所示化合物或式(A-2)所示化合物接触，随后进行分离：
Figure PCTCN2020091484-appb-000066
其中，n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15、L 1、S 1、q 2和R k各自的定义和可选择的范围如前所述。
所述酰胺化反应条件可包括反应温度为0-100℃，反应时间为1-48小时，在一些实施方案中，所述酰胺化反应条件为反应温度为10-40℃，反应时间为2-16小时。
在一些实施方案中，所述有机溶剂为醇类溶剂、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。所述醇类溶剂在一些实施方案中为甲醇、乙醇、丙醇中的一种或多种，在一些实施方案中为乙醇。所述环氧类溶剂在一些实施方案中为为二氧六环和/或四氢呋喃。所述醚类溶剂在一些实施方案中为为乙醚和/或甲基叔丁基醚。所述卤代烷类溶剂在一些实施方案中为为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种。在一些实施方案中，所述有机溶剂为二氯甲烷。相对于式(314)所示化合物，有机溶剂用量为3-50L/mol，在进一步的实施方案中为3-20L/mol。
在一些实施方案中，所述酰胺化反应缩合剂为苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸盐/酯、3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮、4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐、2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ)或O-苯并三氮唑-四甲基脲六氟磷酸盐/酯，在进一步的实施方案中为3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮。所述酰胺化反应缩合剂与式(314)所示化合物的摩尔比可以为1:1-10:1，在一些实施方案中为2.5:1-5:1。
在一些实施方案中，所述三级胺为三乙胺或N,N-二异丙基乙胺，在进一步的实施方案中为N,N-二异丙基乙胺。所述三级胺与式(314)所示化合物的摩尔比为3:1-20:1，在一些实施方案中为5:1-10:1。
在一些实施方案中，式(A-1)和式(A-2)所示化合物可通过任何适当的方式制备。例如，当R k为DMTr基团时，可通过甘油酸钙与DMTrCl反应制备式(A-1)所示化合物；类似地，可先将3-氨基-1,2-丙二醇与环状酸酐接触，随后再与DMTrCl反应制备式(A-2)所示化合物，所述环状酸酐可以是碳原子数为4-13、在一些实施方案中为4-8的环状酸酐。本领域技术人员容易理解的是，所述环状酸酐的选择对应于(A-2)所示化合物中q 2的不同值，例如，当所述环状酸酐为丁二酸酐时，q 2＝1，当所述环状酸酐为戊二酸酐时，q 2＝2，以此类推。
在一些变型中，也可通过使式(314)所示化合物依次与所述环状酸酐、3-氨基-1,2-丙二醇和DMTrCl反应，制备式(313)所示化合物。本领域技术人员容易理解的是，这些变型不会影响式(313)所示化合物的结构与功能，并且这些变型是本领域技术人员在上述方法的基础上容易实现的。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(313)所示化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(313)所示化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用石油醚:乙酸乙酯:二氯甲烷:N,N-二甲基甲酰胺＝1:1:1:0.5-1:1:1:0.6梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇:乙腈＝0.1:1-1:0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(313)所示化合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方案中，式(314)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在酰胺化反应缩合剂和三级胺存在下，在缩合反应条件下，将式(320)所示化合物与式(316)所示化合物接触，随后进行分离：
S 1——L 1——OH
式(316)
Figure PCTCN2020091484-appb-000067
其中，n1、n3、m1、m2、m3、R 10、R 11、R 12、R 13、R 14、R 15各自的定义和可选择的范围如前所述。
式(316)所示化合物可使用例如J.Am.Chem.Soc.2014,136,16958-16961中所公开的化合物，或者，式(316)所示化合物可由本领域技术人员通过各种方法制备，例如，可参照美国专利US 8,106,022 B2实施例1中所公开的方法制备某些式(316)所示化合物，以引用的方式将以上文献的全部内容整体并入本文。
在一些实施方案中，所述缩合反应条件包括反应温度为0-100℃，反应时间为0.1-24小时，在一些实施方案中为反应温度为10-40℃，反应时间为0.5-16小时。
考虑到期望产物式(314)所示化合物的结构，所述式(316)所示化合物与所述式(320)所示化合物的摩尔比应当基于与式(320)中n1与n3的和而确定。在一些实施方案中，例如，当n1+n3＝3时，为了保证反应完全而不过度，式(316)所示化合物与所述式(320)所示化合物的摩尔比可以为3:1-3.5:1，在一些实施方案中为3.01:1-3.15:1。
在一些实施方案中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种，所述环氧类溶剂在一些实施方案中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方案中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方案中为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种，在一些实施方案中，所述有机溶剂为二氯甲烷。相对于式(320)所示化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方案中为5-20L/mol。
在一些实施方案中，所述酰胺化反应缩合剂为苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸盐/酯、3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮(DEPBT)、O-苯并三氮唑-四甲基脲六氟磷酸盐/酯、4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐或1-羟基苯并三唑中的一种或多种，在进一步的实施方案中为苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸盐/酯和1-羟基苯并三唑的混合物，其中苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸酯/盐和1-羟基苯并三唑为等摩尔用量。所述总的酰胺化反应缩合剂与式(316)所示化合物的摩尔比可以为1:1-3:1，在一些实施方案中为1.05:1-1.5:1。
所述三级胺可以为N-甲基吗啉、三乙胺或N,N-二异丙基乙胺，在一些实施方案中为N-甲基吗啉；所述三级胺与式(316)所示化合物的摩尔比可以为2:1-10:1，在一些实施方案中为2:1-5:1。
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(314)所示化合物。在一些实施方案中，可通过蒸发除去溶剂、随后通过色谱方法分离式(314)所示化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用二氯甲烷:甲醇＝100:5-100:7梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇:乙腈＝0.1:1-1:0.1梯度洗脱。在一些实施方案中，可以直接除去溶剂得到式(314)所示化合物粗产品，该粗产品可以直接用于后续反应。
式(320)所示化合物可商购获得，或者由本领域技术人员使用已知的方法获得。例如，当m1＝m2＝m3＝3，n1＝1，n3＝2，且每个R 10、R 11、R 12、R 13、R 14、R 15均为H时，式(320)所示化合物可自阿法埃莎公司商购获得。
本公开的siRNA缀合物也可以与药学上可接受的其它辅料联用，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种，详情可参见上文关于本公开的药物组合物的描述。
本公开的siRNA、含该siRNA的药物组合物及缀合物的应用
在一些实施方案中，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/或预防血栓性疾病和/或缺血性卒中的药物中的用途。
在一些实施方案中，本公开提供了一种预防和/或治疗血栓性疾病和/或缺血性卒中的方法，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/或治疗血栓性疾病和/或缺血性卒中的目的。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/或治疗血栓性疾病和/或缺血性卒中，或用于制备用于预防和/或治疗血栓性疾病和/或缺血性卒中的药物。
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/或治疗血栓性疾病和/或缺血性卒中的手段，在一些实施方案中采用能够将药物递送至肝脏的给药方式。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年一次或多次。
本公开所述的siRNA、药物组合物或siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD 50(使50％的群体死亡的致死剂量)和ED 50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指能引起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/6N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，在一些实施方案中为0.01-50mg/kg体重，在一些实施方案中为0.05-20mg/kg体重，另一些实施方案中为0.1-15mg/kg体重，另一些实施方案中为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，在一些实施方案中为0.01-10mg/kg体重，在一些实施方案中为0.05-5mg/kg体重，在一些实施方案中为0.1-3mg/kg体重。
在一些实施方案中，本公开提供了一种抑制肝细胞中FXI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缀合物与所述肝细胞接触，将本公开的siRNA和/或药物组合物和/或siRNA缀合物导入所述肝细胞，通过RNA干扰的机制达到抑制肝细胞中FXI基因表达的目的。所述肝细胞可以选自SMMC-7721、HepG2、Huh7等肝癌细胞系或分离的肝原代细胞。在一些实施方案中，所述细胞为HepG2肝癌细胞。
采用本公开提供的方法抑制FXI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、0.01nM至100nM、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。
在一些实施方案中，本文所述的试剂盒可在一个容器中提供修饰的siRNA。在一些实施方案中，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。在一些实施方案中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方案中，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。在一些实施方案中，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。在一些实施方案中，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。在一些实施方案中，可在本公开的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor Laboratory Press(1989))所记载的方法进行。
本公开合成的针对FXI基因的siRNA、siRNA缀合物或作为阴性对照的siRNA、siRNA缀合物转染细胞时，使用Lipofectamine TM2000(Invitrogen)作为转染试剂，具体操作参照制造商提供的说明书。
C57BL/6N小鼠：6-8周龄，购自北京维通利华实验动物技术有限公司，以下简称为C57小鼠。
杂合子人源化小鼠：6-8周龄，购自赛业生物科技有限公司。
若无其它说明，以下提供的试剂比例均按体积比(v/v)计算。
若无特别说明，以下体内/体外效果实验数据均以
Figure PCTCN2020091484-appb-000068
表示，数据分析采用Graphpad prism5.0统计分析软件。
制备例1：缀合物L10-siFXIf1M1S的制备
本制备例合成了缀合物L10-siFXIf1M1S。该缀合物为L-9缀合分子与编号为siFXIf1M1S的siRNA缀合后形成的siRNA缀合物。该缀合物中所缀合的siRNA的序列参见表3。
(1-1)L-10化合物的合成
按照以下方法，合成了L-10化合物：
Figure PCTCN2020091484-appb-000069
(1-1-1)缀合末端段GAL-5的合成
Figure PCTCN2020091484-appb-000070
(1-1-1a)GAL-2的合成
将100.0g GAL-1(N-乙酰-D-半乳糖胺盐酸盐，CAS号：1772-03-8，购自宁波弘翔生化公司，463.8mmol)溶于1000ml无水吡啶，冰水浴下加入540ml乙酸酐(购自Enox公司，5565.6mmol)，室温搅拌反应1.5小时。将反应液倒入10L冰水中，减压抽滤，滤饼用2L冰水洗涤后，加乙腈/甲苯混合溶剂(体积比乙腈:甲苯＝1:1)至完全溶解，蒸干溶剂，得到白色固体产品GAL-2 130.0g。
(1-1-1b)GAL-3的合成
将步骤(1-1-1a)中获得的GAL-2(35.1g，90.0mmol)溶解于213ml无水1,2-二氯乙烷中，在冰水浴且氮气保护条件下，加入24.0g TMSOTf(CAS号：27607-77-8，购自麦克林公司，108.0mmol)，室温反应过夜。
在反应液中加入400ml二氯甲烷稀释，以硅藻土过滤，再加入1L饱和碳酸氢钠水溶液，搅拌均匀，分出有机相，水相用二氯乙烷萃取两次，每次300ml，合并有机相，分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到浅黄色粘稠糖稀状产品GAL-3 26.9g。
(1-1-1c)GAL-4的合成
将步骤(1-1-1b)中获得的GAL-3(26.9g，81.7mmol)溶于136ml无水1,2-二氯乙烷中，加入干燥的
Figure PCTCN2020091484-appb-000071
分子筛粉末30g，再加入9.0g 5-己烯-1-醇(CAS号：821-41-0，购自Adamas-beta公司，89.9mmol)，室温下搅拌30分钟，冰浴和氮气保护下加入9.08g TMSOTf(40.9mmol)，室温下搅拌反应过夜。过滤除去
Figure PCTCN2020091484-appb-000072
分子筛粉末，滤液中加入300ml二氯甲烷稀释，以硅藻土过滤，再加入500ml饱和碳酸氢钠水溶液搅拌10分钟洗涤，分出有机相，水相用300ml二氯乙烷萃取一次，合并有机相并分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到黄色糖稀状产品GAL-4 41.3g，不进行纯化直接进行下一步氧化反应。
(1-1-1d)GAL-5的合成
将按照步骤(1-1-1c)中描述的方法得到的GAL-4(14.9g，34.7mmol，)溶于77ml二氯甲烷和77ml乙腈的混合溶剂中，分别加入103ml去离子水和29.7g高碘酸钠(CAS号：7790-28-5，购自阿拉丁公司，138.8mmol)，冰水浴下搅拌10分钟，加入三氯化钌(CAS号：14898-67-0，购自安耐吉公司，238mg，1.145mmol)，室温反应过夜。反应液加入300ml水稀释搅拌，加饱和碳酸氢钠调pH约为7.5，分出并弃去有机相，水相用二氯甲烷萃取三次，每次200ml，弃去有机相。水相用柠檬酸固体调节pH约为3，用二氯甲烷萃取三次，每次200ml，合并有机相，无水硫酸钠干燥，减压蒸干溶剂，得到白色泡沫状固体产品GAL-5 6.85g。 1H NMR(400MHz,DMSO)δ12.01(br,1H),7.83(d,J＝9.2Hz,1H),5.21(d,J＝3.2Hz,1H),4.96(dd,J＝11.2,3.2Hz,1H),4.49(d,J＝8.4Hz,1H),4.07–3.95(m,3H),3.92–3.85(m,1H),3.74–3.67(m,1H),3.48–3.39(m,1H),2.20(t,J＝6.8Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.77(s,3H),1.55–1.45(m,4H).
(1-1-2)L-8的合成
Figure PCTCN2020091484-appb-000073
将J-0(9.886g，52.5mmol，商购自阿法埃沙公司)和步骤(1-1-1)中得到的GAL-5(72.819g，162.75mmol，由多批次产物合并而得)溶于525ml二氯甲烷，加入二异丙基乙胺(DIEA，44.782g，346.50mmol)、苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸酯/盐(PyBOP，90.158g，173.25mmol)和羟基苯并三唑(HOBt，23.410g，173.25mmol)，室温下反应4h，加入20ml饱和碳酸氢钠和200ml饱和食盐水进行洗涤，水相用二氯甲烷萃取2次，每次100ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。纯化使用200-300目正相硅胶，以10wt％三乙胺中和硅胶酸性，1wt‰三乙胺平衡柱子，以二氯甲烷:甲醇＝100:25-100:40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-8 38.8g。 1H NMR(400MHz,DMSO)δ7.84(d,J＝9.0Hz,3H),7.27–7.23(m,1H),7.13–7.18(m,1H),5.22(d,J＝3.1Hz,3H),4.97(dd,J＝11.3,3.1Hz,3H),4.48(d,J＝8.4Hz,3H),4.09–3.98(m,9H),3.88(dd,J＝19.3,9.3Hz,3H),3.75–3.66(m,3H),3.44–3.38(m,3H),3.17–3.30(m,4H),3.10–2.97(m,4H),2.35–2.20(m,6H),2.15–2.08(m,9H),2.07–1.98(m,13H),1.94–1.87(m,9H),1.81–1.74(m,9H),1.65–1.42(m,18H).MS m/z：C 85H 119N 7O 30，[M+H] +，理论：1477.59，实测：1477.23。
(1-1-3)L-7的合成
(1-1-3a)A-1的合成
Figure PCTCN2020091484-appb-000074
将DMTrCl(4,4'-双甲氧基三苯甲基氯，101.65g，300mmol)溶于1000ml无水吡啶中，加入DL-甘油酸钙水合物(28.63g，100mmol)，在45℃反应20h，将反应液过滤，滤饼用200ml DCM淋洗，滤液减压浓缩至干，剩余物用500ml二氯甲烷重新溶解，0.5M三乙胺磷酸盐(pH＝7-8)洗涤2次，每次200ml，水相以二氯甲烷萃取2次，每次200ml，合并有机相，用无水硫酸钠干燥，过滤，减压蒸干溶剂，200-300目正相硅胶柱纯化，以石油醚:乙酸乙酯:二氯甲烷:甲醇＝1:1:1:0.35-1:1:1:0.55梯度洗脱，收集产物洗脱液，减压蒸干溶剂，600ml二氯甲烷重新溶解，以200ml 0.5M三乙胺磷酸盐洗涤1次，水相用200ml二氯甲烷萃取1次，合并有机相，无水硫酸钠干燥，过滤，减压蒸干溶剂，真空油泵减压下过夜，得到白色固体产品A-1 50.7g。 1H NMR(400MHz,DMSO-d6)δ7.46(ddd,J＝6.5,2.3,1.1Hz,1H),7.40–7.28(m,7H),6.89–6.81(m,4H),4.84(d,J＝5.0Hz,1H),4.36–4.24(m,1H),4.29(s,6H),3.92(dd,J＝12.4,7.0Hz,1H),3.67(dd,J＝12.3,7.0Hz,1H),2.52(q,J＝6.3Hz,6H),1.03(t,J＝6.3Hz,9H).MS m/z：C 24H 23O 6，[M-H] -，理论：407.15，实测：406.92。
(1-1-3b)L-7的合成
Figure PCTCN2020091484-appb-000075
将步骤(1-1-2)中获得的L-8(40g，27.09mmol，由多批次产物合并而得)和步骤(1-1-3a)中获得的A-1(41.418g，81.27mmol)混合，溶于271ml二氯甲烷，加入3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮(DEPBT)(24.318g，81.37mmol)，再加入二异丙基乙胺(21.007g，162.54mmol)，25℃下搅拌反应1.5h，用800ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次50ml，以150ml饱和食盐水洗涤有机相，水相以50ml二氯甲烷萃取1次，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，得到粗品。柱纯化使用2kg 200-300目正相硅胶，以200ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚:乙酸乙酯:二氯甲烷:N,N-二甲基甲酰胺＝1:1:1:0.5-1:1:1:0.6梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-7 40.4g。 1H NMR(400MHz,DMSO)δ7.90–7.78(m,4H),7.75–7.64(m,1H),7.38–7.18(m,9H),6.91–6.83(m,4H),5.25–5.10(m,4H),4.97(dd,J＝11.2,3.2Hz,3H),4.48–4.30(m,4H),4.02(s,9H),3.93–3.84(m,3H),3.76–3.66(m,9H),3.45–3.35(m,3H),3.24–2.98(m,10H),2.30–2.20(m,2H),2.11–1.88(m,31H),1.80–1.40(m,28H).MS m/z：C 90H 128N 7O 35，[M-DMTr] +，理论：1564.65，实测：1564.88。
(1-1-4)L-9的合成
Figure PCTCN2020091484-appb-000076
将步骤(1-1-3b)中获得的L-7(40g，21.4247mmol)、丁二酸酐(4.288g，42.8494mmol)和4-二甲氨基吡啶(DMAP，5.235g，42.8494mmol)混合溶于215ml二氯甲烷，再加入二异丙基乙胺(DIEA，13.845g，107.1235mmol)，25℃下搅拌24h，800ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到粗品。柱纯化使用1kg 200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷:甲醇＝100:18-100:20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-9缀合分子31.0g。 1H NMR(400MHz,DMSO)δ8.58(d,J＝4.2Hz,1H),7.94–7.82(m,3H),7.41–7.29(m,5H),7.22(d,J＝8.1Hz,5H),6.89(d,J＝8.3Hz,4H),5.49–5.37(m,1H),5.21(d,J＝3.0Hz,3H),4.97(d,J＝11.1Hz,3H),4.49(d,J＝8.2Hz,3H),4.02(s,9H),3.88(dd,J＝19.4,9.4Hz,3H),3.77–3.65(m,9H),3.50–3.39(m,6H),3.11–2.90(m,5H),2.61–2.54(m,4H),2.47–2.41(m,2H),2.26–2.17(m,2H),2.15–1.95(m,22H),1.92–1.84(m,9H),1.80–1.70(m,10H),1.65–1.35(m,17H),1.31–1.19(m,4H),0.96(t,J＝7.1Hz,9H).MS m/z：C 94H 132N 7O 38，[M-DMTr] +，理论：1664.72，实测：1665.03。
(1-1-5)L-10化合物的合成
Figure PCTCN2020091484-appb-000077
此步骤中，通过将L-9缀合分子连接至固相载体，制备了L-10化合物。
将步骤(1-1-4)中获得的L-9缀合分子(22.751g，11mmol)、O-苯并三氮唑-四甲基脲六氟磷酸盐/酯(HBTU，6.257g，16.5mmol)和二异丙基乙胺(DIEA，2.843g，22mmol)混合，溶于900ml乙腈，室温搅拌5分钟，向反应液中加入氨甲基树脂(88g，100-200目，氨基载量400μmol/g，购自南开和成公司)，25℃下进行摇床反应，转速150转/分钟，反应18h后过滤，滤饼以DCM淋洗2次，每次300ml，乙腈淋洗3次，每次300ml，真空油泵干燥18h，随后再按照表2中示出的投料配比加入原料(CapA、CapB、4-二甲氨基吡啶(DMAP)和乙腈)进行盖帽反应。25℃下置于摇床上，转速150转/分钟，反应5h，反应液过滤，滤饼用乙腈淋洗3次，每次300ml，减压蒸发溶剂至干，真空油泵减压下干燥过夜，得到L-10化合物(即，连接固相载体的L-9缀合分子)102g，载量90.8μmol/g。
表2：盖帽反应投料配比
原料 用量 规格 批号 生产厂家
CapA 1980ml —— —— ——
CapB 220ml —— —— ——
DMAP 1.100g 分析纯 I1422139 Aladdin
乙腈 220ml 光谱纯 O15161001 上海星可
其中，CapA和CapB为盖帽试剂溶液，CapA为20体积％N-甲基咪唑的吡啶/乙腈混合溶液，吡啶与乙腈的体积比为3:5；CapB为20体积％乙酸酐的乙腈溶液。
(1-2)合成缀合物L10-siFXIf1M1S的正义链
通过固相亚磷酰胺法，利用上述步骤制备的L-10化合物起始循环，按照正义链核苷酸排布顺序自3'-5'方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。其中，两个核苷酸之间采用磷酸酯连接时，连接后一个核苷单体时，包括脱保护、偶联、盖帽、氧化四步反应。两个核苷酸之间采用硫代磷酸酯连接时，连接后一个核苷单体时，包括保护、偶联、盖帽、硫化四步反应。合成条件给定如下：
核苷单体以0.1M浓度的乙腈溶液提供，每一步的脱保护反应的条件相同，即温度为25℃，反应时间为70秒，脱保护试剂为二氯乙酸的二氯甲烷溶液(3％v/v)，二氯乙酸与固相载体上4,4'-二甲氧基三苯甲基保护基的摩尔比为5:1。
每一步偶联反应条件均相同，包括温度为25℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1:10，固相载体上连接的核酸序列和偶联试剂的摩尔比为1:65，反应时间为600秒，偶联试剂为5-乙硫基-1H-四氮唑(5-(Ethylthio)-1H-tetrazole，ETT)的0.5M乙腈溶液。
每一步盖帽条件均相同，包括温度为25℃，反应时间为15秒。盖帽试剂溶液为摩尔比为1:1的CapA和CapB的混合溶液，盖帽试剂与固相载体上连接的核酸序列的摩尔比为乙酸酐:N-甲基咪唑:固相载体上连接的核酸序列＝1:1:1。
每一步氧化反应条件相同，包括温度为25℃，反应时间为15秒，氧化试剂为浓度为0.05M的碘水。碘与偶联步骤中固相载体上连接的核酸序列的摩尔比为30:1。反应在四氢呋喃:水:吡啶＝3:1:1的混合溶剂中进行。
每一步硫化反应的条件相同，包括温度为25℃，反应时间为300秒，硫化试剂为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比为120:1。反应在乙腈:吡啶＝1:1的混合溶剂中进行。
待最后一个核苷单体连接完成后，依次对固相载体上连接的核酸序列进行切割、脱保护、纯化、脱盐，随后冻干获得正义链，其中：
切割和脱保护条件如下：将合成的连接有载体的核苷酸序列加入浓度为25wt％的氨水中，氨水用量为0.5ml/μmol，在55℃反应16h，过滤除去剩余载体，将上清液真空浓缩至干。
纯化与脱盐条件如下：利用制备型离子色谱纯化柱(Source 15Q)，通过NaCl的梯度洗脱， 实现核酸的纯化。具体而言为：洗脱剂A：20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9:1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9:1(体积比)；洗脱梯度：洗脱剂A:洗脱剂B＝100:0-50:50梯度洗脱。收集产品洗脱液后合并，采用反相色谱纯化柱进行脱盐，具体条件包括采用葡聚糖凝胶柱进行脱盐，填料为葡聚糖凝胶G25(Sephadex G25)，以去离子水洗脱。
检测方法如下：使用离子交换色谱(IEX-HPLC)检测上述正义链的纯度，使用液质联用(LC-MS)分析分子量。理论值7584.5，实测值7584.0。实测值与理论值相符，表明所合成的是3'末端缀合了L-9缀合分子的正义链SS。
(1-3)合成缀合物L10-siFXIf1M1S的反义链
通过固相亚磷酰胺法，利用通用固相载体(UnyLinker TM loaded
Figure PCTCN2020091484-appb-000078
HL Solid Supports，Kinovate Life Sciences公司)起始循环，合成缀合物L10-siFXIf1M1S的反义链。固相合成方法中的脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同。冻干获得反义链AS。
采用离子交换色谱(IEX-HPLC)进行检测反义链的纯度，采用液质联用(LC-MS)分析反义链的分子量。其结果，实测值与理论值相符，表明所合成的是具有目标序列的反义链AS。
(1-4)合成缀合物L10-siFXIf1M1S
对于缀合物L10-siFXIf1M1S，将正义链与反义链分别溶于注射用水中，得到40mg/mL的溶液，以等摩尔比混合，50℃加热15min，室温冷却后，得到退火后的产品，冻干，得到冻干粉。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将缀合物稀释至浓度为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)进行分子量检测。实测值与理论值一致，说明所合成的siRNA缀合物是目标设计的带有L-9缀合分子的双链核酸序列。其结构如式(403)所示。所述siRNA为表3中所示的对应于缀合物L10-siFXIf1M1S的序列。
表3：siRNA缀合物
Figure PCTCN2020091484-appb-000079
Figure PCTCN2020091484-appb-000080
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；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。
制备例2-10：本公开的siRNA缀合物的合成
按照制备例1的方法，进一步分别合成了以下本公开的siRNA缀合物：L10-siFXIa1M1SP、L10-siFXIb1M1SP、L10-siFXIc1M1SP、L10-siFXId1M1SP、L10-siFXIe1M1SP、L10-siFXIg1M1SP、L10-siFXIh1M1SP、L10-siFXIi1M1S和L10-siFXIi1M1SP。这些缀合物包含的序列分别为表3中的siFXIa1M1SP、siFXIb1M1SP、siFXIc1M1SP、siFXId1M1SP、siFXIe1M1SP、siFXIg1M1SP、siFXIh1M1SP、siFXIi1M1S和siFXIi1M1SP。制备方法的区别仅在于：(1)分别以表3中各缀合物所包含的siRNA正义链和反义链序列代替缀合物L10-siFXIf1M1S的正义链和反义链序列；(2)对于缀合物L10-siFXIa1M1SP、L10-siFXIb1M1SP、L10-siFXIc1M1SP、L10-siFXId1M1SP、L10-siFXIe1M1SP、L10-siFXIg1M1SP、L10-siFXIh1M1SP和L10-siFXIi1M1SP，它们的反义链的5'-末端第一个核苷酸为5'-磷酸核苷酸，相应地，在按照固相亚磷酰胺法制备反义链的过程中，连接反义链最后一个核苷单体后，再经脱保护、偶联、盖帽、氧化四步反应将CPR-I单体(苏州吉玛，货号Cat#13-2601-XX)连接至反义链5'末端，形成5'-磷酸核苷酸。
Figure PCTCN2020091484-appb-000081
该连接中，使用的脱保护、偶联、盖帽、氧化反应条件合成正义链相同。该连接完成后，再进行切割与脱保护、纯化与脱盐步骤，最后冻干获得反义链AS。
制备完成后，按照制备例1的方法对所制备得到的各缀合物的分子量分别进行检测，实测值与理论值一致，说明所合成的siRNA缀合物是目标设计的带有L-9缀合分子的双链核酸序列。其结构均如式(403)所示。这些缀合物所包含的siRNA分别为表3中所示的对应于缀合物L10-siFXIa1M1SP、L10-siFXIb1M1SP、L10-siFXIc1M1SP、L10-siFXId1M1SP、L10-siFXIe1M1SP、L10-siFXIg1M1SP、L10-siFXIh1M1SP、L10-siFXIi1M1S或L10-siFXIi1M1SP的序列。
制备例11-20：本公开提供的siRNA的合成
通过固相合成方法分别合成表4中所列的siRNA序列，并进行分子量检测。使用DEPC水，分别溶解等摩尔表4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以下siRNA：siFXIa1M1SP、siFXIb1M1SP、siFXIc1M1SP、siFXId1M1SP、siFXIe1M1SP、siFXIf1M1SP、siFXIg1M1SP、siFXIh1M1SP、siFXIi1M1SP以及siFXIe1，如表4所示。
在序列siFXIe1的制备过程中，目标序列中包含未修饰的核苷酸。此时，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。
并且，对于目标序列中反义链5'-末端第一个核苷酸为5'-磷酸核苷酸的情况，在按照固相亚磷酰胺法制备反义链的过程中，在连接反义链最后一个核苷单体后，再经脱保护、偶联、盖帽、氧化四步反应将CPR-I单体(苏州吉玛，货号Cat#13-2601-XX)连接至反义链5'末端，形成5'- 磷酸核苷酸。
Figure PCTCN2020091484-appb-000082
该连接中，使用的脱保护、偶联、盖帽、氧化反应条件与合成正义链相同。该连接完成后，再进行切割与脱保护、纯化与脱盐步骤，最后冻干获得反义链AS。
对比制备例1：参比siRNA的合成
通过固相合成方法分别合成了表4中siRNA编号为NC的siRNA对应的正义链和反义链，并进行分子量检测。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。
表4：siRNA序列
Figure PCTCN2020091484-appb-000083
Figure PCTCN2020091484-appb-000084
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；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。
在上述本公开的siRNA或缀合物制备完成后，冻干为固体粉末保存备用。在使用时，可使用例如注射用水、生理盐水(NS)、磷酸缓冲液(PB)或者磷酸盐缓冲液(PBS)等将其重新溶解为所需浓度的溶液使用。
实验例1：本公开的siRNA体外(in vitro)的抑制活性
用含有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DMEM完全培养基(Hyclone公司)于37℃在含5％CO 2/95％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，将待评价的siRNA(siFXIe1)转染至HEK293A细胞中，通过双萤光素酶报告基因的表达水平，来反应siRNA的抑制活性。具体步骤如下：
[1]构建检测质粒
采用psiCHECK TM-2(Promega TM)质粒构建检测质粒，该质粒含有一个目标序列，即siRNA靶序列。对于待测siRNA，目标序列如下所示：
siFXIe1(由制备例20制备获得)的目标序列为：
GAATCTCAAAGAAATCTTT(SEQ ID NO:565)
将目标序列克隆到psiCHECK TM-2质粒的Xho I/Not I位点。
[2]转染
将HEK293A细胞以8×10 3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。
用DEPC化水将上述检测质粒稀释成200ng/μl的检测质粒工作液；用siFXIe1和DEPC化水配制成浓度分别为10nM和3nM(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10nM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3nM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。
分别将一份1B溶液与一份1A1溶液或一份1A2溶液混合，室温下孵育20min，分别得到转染复合物1X1、1X2，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1X3。
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0.1nM的共转染混合物，记为测试组1。
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0.03nM的共转染混合物，记为测试组2。
在另外三个培养孔中，分别加入转染复合物1X3，得到不含siRNA的转染混合物，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24h。
[3]检测
吸去培养孔中的培养基，每孔加入150μl的
Figure PCTCN2020091484-appb-000085
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 PCTCN2020091484-appb-000086
Stop&
Figure PCTCN2020091484-appb-000087
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siFXIe1对目标序列的抑制活性结果如表5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。结果如表5所示。
表5：目标序列的抑制率
Figure PCTCN2020091484-appb-000088
结果表明，siFXIe1在各浓度下对目标序列均具有较好的体外抑制活性，且呈现出浓度依赖性的抑制活性。特别是，在0.1nM的siRNA浓度下，目标序列抑制率为72.43％，显示出良好的抑制FXI基因表达的效果。
实验例2：siRNA序列在psiCHECK系统中对FXI mRNA的IC 50测定
本实验例考察了siFXIa1M1SP、siFXIb1M1SP、siFXIc1M1SP、siFXId1M1SP、siFXIe1M1SP和siFXIi1M1SP在体外psiCHECK系统中的IC 50值。
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，将检测质粒与待测siRNA共转染至HepG2细胞中，通过双萤光素酶报告基因的表达水平，来反映siRNA的在靶活性及脱靶效应。具体步骤如下：
[1]构建检测质粒
采用psiCHECK TM-2(Promega TM)质粒构建了检测质粒，所述质粒中含有一个目标序列，该目标序列具有Genbank注册号为NM_00128.3所示的序列。
将目标序列克隆到psiCHECK TM-2质粒的Xho I/Not I位点。
[2]细胞培养及转染
HepG2细胞购自广州吉妮欧生物科技有限公司，用含有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DMEM完全培养基(HyClone公司)培养细胞，于37℃在含5％CO 2/95％空气的培养箱中培养。
将HepG2细胞以8×10 3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。
用DEPC化水将上述检测质粒稀释成200ng/μl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、33.3nM、11.1nM、3.70nM、1.23nM、4.12nM、0.137nM、0.0457nM、0.0152nM和0.00508nM共10种不同浓度的siRNA工作液，所用siRNA分别为siFXIa1M1SP、siFXIb1M1SP、siFXIc1M1SP、siFXId1M1SP、siFXIe1M1SP和siFXIi1M1SP。
对于每一个siRNA,分别配制2A1-2A10溶液，每份2A1-2A10溶液依次含有上述10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。
分别将一份1B溶液与得到的一份每个siRNA的2A1-2A10的溶液混合，分别室温下孵育20min，得到每个siRNA的转染复合物2X1-2X10。
在培养孔中，分别加入每一个siRNA转染复合物2X1-2X10，均匀混合，加入量为20μl/孔，得到每个siRNA终浓度分别约为1nM、0.333nM、0.111nM、0.0370nM、0.0123nM、0.00412nM、0.00137nM、0.000457nM、0.000152nM和0.0000508nM的转染复合物，每个siRNA的转染复 合物2X1-2X10分别转染3个培养孔，得到含siRNA的共转染混合物，记为测试组。
在另外三个培养孔中，分别加入转染复合物1X3，加入量为20μl/孔，得到不含siRNA的共转染混合物，记为对照组。
分别将含siRNA的共转染混合物和不含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24h。
[3]检测
吸去培养孔中的培养基，每孔加入150μl的
Figure PCTCN2020091484-appb-000089
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 PCTCN2020091484-appb-000090
Stop&
Figure PCTCN2020091484-appb-000091
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的抑制率为(1-R)×100％。
利用Graphpad 5.0软件log(inhibitor)vs.response—Variable slope功能来拟合剂量-效应曲线，根据剂量-效应曲线计算待测siRNA靶向GSCM的IC 50值。具体来说，拟合获得的剂量效应曲线符合以下公式：
Figure PCTCN2020091484-appb-000092
式中：
Y是比值R，即残留活性，
X为转染siRNA浓度的对数值，
Bot是稳态期底部的Y值，
Top是稳态期顶部的Y值，
X'是拟合获得的当Y在底部到顶部之间一半时的X值，而HillSlope则是拟合获得的曲线在X'处的斜率。
由该剂量-效应曲线和对应的计算公式，确定当Y＝50％时对应的X 50值，计算获得各siRNA的IC 50值＝10^X 50。
具体IC 50值总结于表6中。
表6：siRNA的IC 50
制备例编号 siRNA编号 IC 50
制备例11 siFXIa1M1SP 0.024nM
制备例12 siFXIb1M1SP 0.078nM
制备例13 siFXIc1M1SP 0.119nM
制备例14 siFXId1M1SP 0.071nM
制备例15 siFXIe1M1SP 0.013nM
制备例19 siFXIi1M1SP 0.041nM
由上述表6的结果可知，本公开提供的siRNA在体外HepG2细胞中对目标序列1有很高的抑制活性，IC 50在0.013-0.119nM之间。
实验例3：siRNA在HepG2细胞中对FXI mRNA的IC 50检测
将HepG2细胞以7×10 4细胞/孔接种于24孔板中，16h后细胞生长密度达到70-80％时，吸尽培养孔中H-DMEM完全培养基，每孔加入500μl Opti-MEM培养基(GIBCO公司)继续培养1.5h。
用DEPC化水将下面的siRNA中的每一个siRNA分别配制成20μM、6.67μM、2.22μM、0.741μM、0.247μM、0.0823μM和0.0274μM的共7种不同浓度的siRNA工作液，所用siRNA分别为siFXIa1M1SP、siFXIb1M1SP、siFXIc1M1SP或siFXId1M1SP。
配制3A1-3A7溶液，对于每一个siRNA,分别配制3A1-3A7溶液，每份3A1-3A7溶液依次含 有上述7个浓度的siRNA工作液3μl和Opti-MEM培养基50μl。
配制3B溶液，每份3B溶液含有1μl Lipofectamine TM 2000和50μl Opti-MEM培养基。
分别将一份3B溶液与得到的一份每个siRNA的3A1-3A7的溶液混合，分别在室温下孵育20min，得到每个siRNA的转染复合物3X1-3X7。
将一份3B溶液与Opti-MEM培养基50μl混合，室温下孵育20min，得到转染复合物3X8。
在培养孔中，分别加入每一个siRNA转染复合物3X1-3X7，均匀混合，加入量为100μl/孔，得到每个siRNA终浓度分别约为100nM、33.3nM、11.1nM、3.70nM、1.23nM、0.412nM、0.137nM的转染化混合物，每个siRNA的转染复合物3X1-3X7分别转染3个培养孔，得到含siRNA的转染混合物，记为测试组。
在另外三个培养孔中，分别加入转染复合物3X8，加入量为100μl/孔，得到不含siRNA的转染混合物，记为对照组。
将含siRNA的转染混合物和不含siRNA的转染混合物在培养孔中培养4h后，每孔补加1ml含20％FBS的H-DMEM完全培养基。将24孔板置于CO 2培养箱继续培养24h。
随后，使用RNAVzol(购自威格拉斯生物技术(北京)有限公司，货号N002)根据说明书描述的详细步骤提取各孔细胞中的总RNA。
对于每孔细胞，分别取1μg总RNA，使用反转录试剂盒Goldenstar TM RT6 cDNA Synthesis Kit(购自北京擎科新业生物技术有限公司，货号TSK301M)提供的试剂，其中选取Goldenstar TM Oligo(dT) 17作为引物，按试剂盒说明书中反转录操作步骤配置反转录反应体系20μl，对细胞的总RNA进行反转录。反转录的条件为：将反转录反应体系置于50℃孵育50min，然后85℃孵育5min，最后4℃孵育30s，反应结束后，向反转录反应体系中加入DEPC水80μl，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μl做模板，使用
Figure PCTCN2020091484-appb-000093
SYBR qPCR SuperMix Plus试剂盒(购自近岸蛋白质科技有限公司，货号E096-01B)提供的试剂配置qPCR反应体系20μl，其中，用于扩增目标基因FXI和内参基因GAPDH的PCR引物序列如表7所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火30s，72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因FXI和内参基因GAPDH的产物W。产物W随即依次经过95℃ 15s，60℃ 1min，95℃ 15s的孵育，实时荧光定量PCR仪分别收集产物W中目标基因FXI和内参基因GAPDH的溶解曲线，得到目标基因FXI和内参基因GAPDH的Ct值。
表7：检测引物的序列
Figure PCTCN2020091484-appb-000094
采用比较Ct(ΔΔCt)法，对各测试组和对照组中目标基因FXI的表达量进行相对定量计算，计算方法如下：
ΔCt(测试组)＝Ct(测试组目标基因)–Ct(测试组内参基因)
ΔCt(对照组)＝Ct(对照组目标基因)–Ct(对照组内参基因)
ΔΔCt(测试组)＝ΔCt(测试组)-ΔCt(对照组平均)
ΔΔCt(对照组)＝ΔCt(对照组)-ΔCt(对照组平均)
其中，ΔCt(对照组平均)是对照组三个培养孔各自的ΔCt(对照组)的算术平均值。从而，测试组和对照组的每一培养孔均对应一个ΔΔCt值。
以对照组为基准，对测试组FXI mRNA的表达水平进行归一化，定义对照组FXI mRNA表达水平为100％，
测试组FXI mRNA相对表达水平＝2 -ΔΔCt(测试组)×100％。
对于同一测试组siRNA，每一浓度下的测试组FXI mRNA相对表达水平平均值为该浓度三个培养孔的相对表达水平的算术平均值。
利用Graphpad 5.0软件的非线性回归分析功能拟合log(inhibitor)vs.response—Variable slope剂量-效应曲线，根据剂量-效应曲线计算各siRNA对FXI mRNA的IC 50值。具体来说，拟合获得的剂量-效应曲线符合以下计算公式：
Figure PCTCN2020091484-appb-000095
式中：
Y是各测试组FXI mRNA相对表达水平，
X为对应测试组所使用的siRNA终浓度的对数值，
Bot是稳态期底部的Y值，
Top是稳态期顶部的Y值，
X'是拟合获得的当Y在底部到顶部之间一半时的X值，而HillSlope则是拟合获得的曲线在X'处的斜率。
由该剂量-效应曲线和对应的计算公式，确定当Y＝50％时对应的X 50值，计算获得各siRNA的IC 50值＝10^X 50(nM)。
各siRNA对FXI mRNA的IC 50值总结于表8中。
表8：siRNA对FXI mRNA的IC 50
制备例编号 编号 IC 50
制备例2 siFXIa1M1SP 6.18nM
制备例3 siFXIb1M1SP 7.54nM
制备例4 siFXIc1M1SP 11.1nM
制备例5 siFXId1M1SP 1.49nM
由表8可见，本公开提供的siRNA在体外HepG2细胞系中具有很高的抑制FXI mRNA的活性，IC 50在1.49-11.1nM之间。
实验例4：siRNA在小鼠肝原代细胞中对FXI mRNA的IC 50检测
小鼠肝原代细胞由正常C57BL/6N小鼠新鲜肝组织提取获得，在I型胶原蛋白包被的玻璃或塑料盖玻片或组织培养皿中接种适当密度的细胞，用含有1×双抗和10％FBS的RPMI 1460培养基培养细胞，于37℃在含5％CO 2/95％空气的培养箱中培养细胞30min。
按照实验例3的方法，测定siRNA对FXI mRNA的抑制活性和IC 50值，区别仅在于，所检查的siRNA为siFXIf1M1SP；所使用的细胞为小鼠肝原代细胞；siRNA终浓度分别为100nM、25nM、6.25nM、1.56nM、0.391nM、0.098nM、0.0244nM和6.1×10 -3nM，共8个浓度。结果如表9所示。
表9：siRNA对FXI mRNA的IC 50
制备例编号 编号 IC 50
制备例7 siFXIf1M1SP 0.021nM
由表9可见，siFXIf1M1SP在体外小鼠肝原代细胞中具有很高的抑制FXI mRNA的活性，IC 50为0.021nM。
实验例5：siRNA在HepG2细胞中对FXI mRNA表达量的抑制效率检测。
按照实验例3的方法，测定了siRNA对FXI mRNA的表达量抑制率，区别仅在于，所用siRNA分别为siFXIg1M1SP和siFXIh1M1SP；对于每一siRNA，siRNA终浓度分别为50nM、5nM和0.5nM，共3个浓度，每个浓度2个培养孔，结果如表10所示。
表10：不同浓度siRNA的FXI mRNA抑制率
Figure PCTCN2020091484-appb-000096
由表10可见，本公开提供的siRNA在体外HepG2细胞中具有很高的抑制活性，在siRNA浓度为50nM时可达到83％的FXI mRNA抑制率。
实验例6：缀合物L10-siFXIf1M1S、L10-siFXIi1M1S和L10-siFXIi1M1SP在小鼠体内对FXI mRNA表达的抑制
将C57BL/6N小鼠随机分组(均为雌性)，每组5只小鼠，分别进行编号。以皮下注射的方式，对每组小鼠分别以5mg/kg以及1mg/kg(均以siRNA计)的不同剂量给予待测缀合物L10-siFXIf1M1S、L10-siFXIi1M1S或L10-siFXIi1M1SP。siRNA缀合物分别以1mg/ml以及0.2mg/ml的siRNA缀合物的0.9％氯化钠水溶液形式提供，给药体积均为5ml/kg。
对其中一组小鼠给与1×PBS，给药体积均为5ml/kg，作为对照组。
给药后第7天处死动物，分别收集每只小鼠的肝脏组织，用RNA later(Sigma Aldrich公司)保存；用组织匀浆仪匀浆肝组织，再用Trizol(Thermo Fisher公司)根据说明书描述的操作步骤提取得到总RNA。
按照实验例3的方法进行荧光定量PCR检测和计算FXI mRNA的表达水平及抑制率，区别 仅在于：使用ImProm-IITM反转录试剂盒(Promega公司)按其说明书将提取的总RNA逆转录为cDNA，得到含cDNA的溶液，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)检测肝组织中的FXI mRNA的表达水平。在该荧光定量PCR法中，以鼠GAPDH(mGAPDH)基因作为内参基因，使用针对FXI的引物和针对鼠GAPDH的引物分别对FXI和鼠GAPDH进行检测。检测引物的序列参见表11所示。在FXI mRNA表达水平及抑制率的计算中，对照组为本实验中施以PBS的对照组小鼠，各测试组为分别施以不同siRNA缀合物的给药组小鼠。对照组的FXI mRNA表达水平记为100％，相应地，FXI mRNA表达水平抑制率记为0％，测试结果以对照组的FXI mRNA表达水平进行标准化，结果示于表12中。
表11：检测引物的序列
Figure PCTCN2020091484-appb-000097
表12：不同浓度siRNA缀合物的FXI mRNA抑制率
Figure PCTCN2020091484-appb-000098
由表12的结果可见，本公开的siRNA缀合物在1mg/kg的siRNA给药剂量下就显示出56.8-78.4％的FXI mRNA抑制率；在5mg/kg的siRNA浓度下，FXI mRNA抑制率更是高达95.0％，显示出优异的FXI mRNA的抑制效果。
实验例7：
缀合物L10-siFXIf1M1S和L10-siFXIi1M1SP在小鼠体内给药后不同时间点对FXI mRNA表达的抑制以及活化部分凝血活(APTT)的延长的测定
将C57BL/6N小鼠随机分为7组(均为雄性)，每组5只小鼠，分别进行编号。对其中的各三组小鼠分别给与缀合物L10-siFXIf1M1S和L10-siFXIi1M1SP。对其中的一组小鼠给与生理盐水(Saline)，作为对照组。给药方式采用皮下给药的方式，缀合物以1.8mg/ml(以siRNA计)的siRNA缀合物的0.9％氯化钠水溶液形式提供，生理盐水为0.9％氯化钠水溶液，给药体积均为5ml/k，缀合物给药量为9mg/kg。分别在给药后第8天、第15天或第29天收集血浆，给药组在给药后第29天处死动物，对照组在给药第8天处死动物。分别收集每只小鼠的肝脏，用RNA later(Sigma Aldrich公司)保存，用组织匀浆仪匀浆肝组织，再用Trizol(Thermo Fisher公司)根据说明书描述的操作步骤提取得到总RNA。
按照实验例3的方法对每只小鼠的FXI mRNA的表达量进行荧光定量PCR检测和计算FXI mRNA的表达水平及抑制率，区别仅在于：使用ImProm-IITM反转录试剂盒(Promega公司)按其说明书将提取的总RNA逆转录为cDNA，得到含cDNA的溶液，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)检测肝组织中的FXI mRNA的表达水平。在该荧光定量PCR法中，以鼠GAPDH(mGAPDH)基因作为内参基因，使用针对FXI的引物和针对鼠GAPDH的引物分别对FXI和鼠GAPDH进行检测。检测引物的序列参见表11所示。
在FXI mRNA表达量及抑制率的计算中，对照组为本实验中施以生理盐水的对照组小鼠，各测试组为分别施以不同siRNA缀合物并在不同给药时间后取样的给药组小鼠。对照组的FXI mRNA表达水平记为100％，相应地，FXI mRNA表达水平抑制率记为0％，测试结果以对照组的FXI mRNA表达水平进行标准化，结果示于表13中。表13中，FXI mRNA表达水平抑制率为给与相对应的siRNA缀合物相对应天数后的，该组内5只小鼠FXI mRNA表达量抑制率的算术平均值。
表13：单次给药后不同时间点siRNA缀合物的FXI mRNA抑制率
Figure PCTCN2020091484-appb-000099
Figure PCTCN2020091484-appb-000100
由表13的结果可见，在小鼠皮下单次给药后，本公开的siRNA缀合物在长时间内的不同时间点均显示出优异的肝内FXI mRNA抑制率，抑制率至少为89.18％，甚至高达92.89％。
进一步地，对于上述收集的血浆，使用APTT试剂盒(Rayto公司，批号20190402M)，在半自动凝血仪(Rayto公司，型号RT-2202)上以比浊法测定每只小鼠的血浆APTT值，具体测定方法依照APTT试剂盒的说明书描述的方法进行。测定的APTT与对照组进行对比，每只小鼠APTT相对延长时间＝(测试组APTT检测值-对照组APTT检测平均值)/(对照组APTT检测平均值)×100％。测定结果参见表14所示。表14中，APTT相对延长时间为给与相对应的siRNA缀合物相对应天数后的，该组内5只小鼠APTT相对延长时间的平均值。
表14：单次给药后不同时间点siRNA缀合物的APTT相对延长时间
Figure PCTCN2020091484-appb-000101
由表14的结果可见，在给予本公开的siRNA缀合物后，小鼠在较长时间内均显示出显著延长的APTT检测值，最高可延长64.9％。可见，本公开的siRNA缀合物能够有效延长小鼠凝血时间，表明其具有优异的治疗和/或预防血栓性疾病和缺血性卒中的应用前景。
实验例8：本公开提供的siRNA缀合物在人源化小鼠体内(in vivo)活性的测定
本试验例中使用的人源化小鼠购自赛业生物科技有限公司，将人源化小鼠随机分组，每组4只(2雄2雌)，分别向每组小鼠给予缀合物L10-siFXIf1M1S、L10-siFXIa1M1SP、L10-siFXIb1M1SP、L10-siFXIc1M1SP、L10-siFXId1M1SP、L10-siFXIe1M1SP、L10-siFXIg1M1SP、L10-siFXIh1M1SP和L10-siFXIi1M1S和生理盐水(saline)对照。所有动物根据体重计算药量，采用皮下注射方式单次给药，各缀合物以0.3mg/ml(以siRNA计)的0.9％氯化钠水溶液形式给予，给药体积为10ml/kg小鼠体重，即，每一缀合物的给药剂量为3mg/kg(以siRNA计)。给药后第8天，处死小鼠，收集血浆，以抗凝剂与血浆的体积比1:9(v/v)的比例加入3.2wt％(0.109mol/L)二水合柠檬酸钠水溶液防止凝血，离心分离血浆。
取肝脏左大叶约100mg/鼠，用RNA later(Sigma Aldrich公司)保存；随后对于每只小鼠的肝脏组织，分别用组织匀浆仪匀浆肝组织，再用Trizol(Thermo Fisher公司)根据说明书描述的操作步骤提取得到每只小鼠的肝组织总RNA。
按照实验例6的方法，通过实时荧光定量PCR法对给予不同的本公开的siRNA缀合物的小鼠以及对照组小鼠肝组织中FXI mRNA的表达水平进行检测，区别仅在于，用于扩增人FXI和作为内参基因的鼠GAPDH的引物序列如表15所示。
表15：检测引物的序列
Figure PCTCN2020091484-appb-000102
按照实验例3的方法测定的FXI mRNA表达水平并计算FXI mRNA抑制率，对照组的FXI mRNA表达水平记为100％，相应地，FXI mRNA表达水平抑制率记为0％，测试结果以对照组的FXI mRNA表达水平进行标准化，结果示于表16中。表16中，人FXI mRNA抑制率为给与相对应的siRNA缀合物的一组小鼠人FXI mRNA抑制率的平均值及其标准偏差。
表16：本公开的siRNA缀合物在人源化小鼠体内对人FXI mRNA的抑制率
制备例编号 缀合物编号 人FXI mRNA抑制率％
制备例1 L10-siFXIf1M1S 76.03±6.74
制备例2 L10-siFXIa1M1SP 89.23±3.25
制备例3 L10-siFXIb1M1SP 81.75±3.91
制备例4 L10-siFXIc1M1SP 81.25±3.61
制备例5 L10-siFXId1M1SP 71.06±9.62
制备例6 L10-siFXIe1M1SP 85.26±4.15
制备例7 L10-siFXIg1M1SP 93.09±1.96
制备例8 L10-siFXIh1M1SP 76.78±5.54
制备例9 L10-siFXIi1M1S 74.25±6.07
由表16的结果可见，本公开的siRNA在人源化杂合子小鼠肝中对人FXI mRNA均显示出较好的抑制效果，对FXI mRNA的抑制率高达约71-93％左右。
进一步地，使用Human Coagulation Factor X ELISA试剂盒(Sigma公司，批号0926F2350，货号RAB1385-1KT)测试自上述各组小鼠(分别给予缀合物L10-siFXIf1M1S、L10-siFXIa1M1SP、L10-siFXIb1M1SP、L10-siFXIc1M1SP、L10-siFXId1M1SP、L10-siFXIe1M1SP、L10-siFXIg1M1SP、L10-siFXIh1M1SP或L10-siFXIi1M1S的测试组和盐水(saline)对照组)获取的血浆中的FXI蛋白含量。
用去离子水将ELISA试剂盒中的样品稀释液(试剂盒中标注为ItemE2，)进行5倍稀释，得到稀释后的样品稀释液。
对于给予缀合物L10-siFXIa1M1SP或L10-siFXIg1M1SP的小鼠血浆，向12μL血浆中加入108μL稀释后的样品稀释液，获得待测样品液，留存备用。
对于给予其它缀合物或盐水的小鼠血浆，分别向12μL血浆中加入108μL稀释后的样品稀释液，获得10倍稀释血浆。向5μL该10倍稀释血浆中加入45μL稀释后的样品稀释液，获得100倍稀释血浆；再向12μL该100倍稀释血浆中加入108μL稀释后的样品稀释液，获得稀释1000倍的样品稀释液，作为待测样品液留存备用。
以100μL稀释后的样品稀释液将试剂盒中的FXI检测抗体(试剂盒中标注为ItemF)溶解为抗体样品，随后取75μL该抗体样品加入5925μL稀释后的样品稀释液进行80倍稀释，获得检测抗体溶液。
以稀释后的样品稀释液将试剂盒中的链霉素浓缩液(试剂盒中标注为ItemG)稀释250倍，获得链霉素稀释液。
以去离子水将试剂盒中的洗涤缓冲液(试剂盒中标注为ItemB)稀释20倍，获得稀释洗涤液。
提供8种标准浓度梯度溶液，其中一种是稀释后的样品稀释液(可视为0pg/mL浓度的标准品溶液)，另外7种是将试剂盒中的标准品(试剂盒中标注为ItemC)用前述得到的稀释后的样品稀释液依次稀释，得到的浓度为2500pg/mL、1000pg/mL、400pg/mL、160pg/mL、64pg/mL、25.6pg/mL和10.24pg/mL，共7个浓度值的标准品溶液。
ELISA检测
使用Human Coagulation Factor X ELISA试剂盒(SIGMA公司，货号RAB1385-1KT)，根据使用说明书设置标准品孔和样本孔，在每孔中分别加入不同浓度的标准浓度梯度溶液或者各待测样品液，每孔加入溶液各100μL，室温孵育2.5小时。弃去液体，每孔加入300μL稀释洗涤液洗涤1min后弃去洗涤液。在每孔中加入100μL检测抗体溶液，室温下孵育1小时。弃去液体，每孔加入300μL稀释洗涤液洗涤1min后弃去洗涤液，再重复按此步骤洗涤3次(共洗涤4次)。在每孔中加入100μL链霉素稀释液，室温孵育45分钟。弃去液体，每孔加入300μL稀释洗涤液洗涤1min后弃去洗涤液，再重复按此步骤洗涤3次(共洗涤4次)。在每孔中加入100μLTMB(试剂盒中标注为ItemH)，孵育30分钟。在每孔中加入终止液(试剂盒中提供)50μL终止反应，立即用全自动酶标仪(BioTek公司，Biotck SYNERGY MX)在450nm波长处读取波长。每一特定浓度的测试组以盐水处理为对照。
根据对标准浓度梯度溶液所测得的活性结果，GraphPad Prism 6软件log(inhibitor)vs.response—Variable slope功能来拟合剂量-效应标准曲线，根据剂量-效应曲线计算待测血浆中蛋白浓度值，拟合获得的剂量-效应曲线符合以下计算公式：
Figure PCTCN2020091484-appb-000103
式中：
Y是在450nm处读取的相应光密度值，
X为标准曲线中浓度的对数值(μg/mL)，
Bot是稳态期底部的Y值，
Top是稳态期顶部的Y值，
X'是拟合获得的当Y在底部到顶部之间一半时的X值，而HillSlope则是X'处曲线的斜率，
由获得的标准曲线，代入各血浆样品测定的光密度值，获得各样品对应的浓度对数值X，计算获得各给予不同siRNA缀合物的样品的血浆FXI蛋白浓度值＝10^X(μg/mL)。
根据血浆FXI蛋白浓度值，相对于对照组的蛋白浓度，计算FXI蛋白抑制率＝(对照组蛋白浓度-测试组蛋白浓度)/对照组蛋白浓度×100％。所得的浓度结果和抑制率数据示于表17中。表17中，FXI蛋白浓度和FXI蛋白抑制率分别为给与相对应的siRNA缀合物的一组小鼠FXI蛋白浓度和FXI蛋白抑制率的算术平均值。
表17：本公开的siRNA缀合物对血浆中蛋白浓度表达效果的抑制
制备例编号 缀合物编号 FXI蛋白浓度(μg/mL) FXI蛋白相对抑制率％
对照组 (盐水) 0.2597 0
制备例1 L10-siFXIf1M1S 0.0469 81.93
制备例2 L10-siFXIa1M1SP 0.0026 99.02
制备例3 L10-siFXIb1M1SP 0.0258 90.08
制备例4 L10-siFXIc1M1SP 0.0205 92.09
制备例5 L10-siFXId1M1SP 0.0417 83.94
制备例6 L10-siFXIe1M1SP 0.0166 93.61
制备例7 L10-siFXIg1M1SP 0.0017 99.34
制备例8 L10-siFXIh1M1SP 0.0447 82.80
制备例9 L10-siFXIi1M1S 0.0533 79.47
由表16的结果可见，本公开的siRNA缀合物在人源化杂合子小鼠血浆中均显示出非常优异的抑制人FXI蛋白的表达的效果，特别是缀合物L10-siFXIa1M1SP和L10-siFXIg1M1SP均显示出99％左右的高FXI蛋白抑制率。
以上详细描述了本公开的一些实施方案，但是，本公开并不限于上述实施方案中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述一些实施方案中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方案之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。
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。

Claims (71)

  1. 一种siRNA，所述siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，所述核苷酸序列I和所述核苷酸序列II选自如下i)-ix)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'-GGGUAUUCUUUCAAGCAAZ 1-3'(SEQ ID NO:1)；
    5'-Z 2UUGCUUGAAAGAAUACCC-3'(SEQ ID NO:2)，
    其中，Z 1为U，Z 2为A，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'-GGCAUAAACUAUAACAGCZ 5-3'(SEQ ID NO:61)；
    5'-Z 6GCUGUUAUAGUUUAUGCC-3'(SEQ ID NO:62)，
    其中，Z 5为U，Z 6为A，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'-GCUCAAGAAUGCCAAGAAZ 9-3'(SEQ ID NO:121)；
    5'-Z 10UUCUUGGCAUUCUUGAGC-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；
    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'-GCAACAAAGACAUUUAUGZ 13-3'(SEQ ID NO:181)；
    5'-Z 14CAUAAAUGUCUUUGUUGC-3'(SEQ ID NO:182)，
    其中，Z 13为U，Z 14为A，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'-GAAUCUCAAAGAAAUCUUZ 17-3'(SEQ ID NO:241)；
    5'-Z 18AAGAUUUCUUUGAGAUUC-3'(SEQ ID NO:242)，
    其中，Z 17为U，Z 18为A，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'-GUACGUGGACUGGAUUCUZ 21-3'(SEQ ID NO:301)；
    5'-Z 22AGAAUCCAGUCCACGUAC-3'(SEQ ID NO:302)，
    其中，Z 21为G，Z 22为C，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'-AUUUCUGGGUAUUCUUUCZ 25-3'(SEQ ID NO:361)；
    5'-Z 26GAAAGAAUACCCAGAAAU-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'-CAUGAAGGGCAUAAACUAZ 29-3'(SEQ ID NO:421)；
    5'-Z 30UAGUUUAUGCCCUUCAUG-3'(SEQ ID NO:422)，
    其中，Z 29为U，Z 30为A，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；以及
    ix)所述核苷酸序列I与SEQ ID NO:481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:482所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GGAUUCUGGAGAAAACUCZ 33-3'(SEQ ID NO:481)；
    5'-Z 34GAGUUUUCUCCAGAAUCC-3'(SEQ ID NO:482)，
    其中，Z 33为A，Z 34为U，所述核苷酸序列I中包含位置对应于Z 33的核苷酸Z 35，所述核苷酸序列II中包含位置对应于Z 34的核苷酸Z 36，所述Z 36是所述反义链5'末端的第一个核苷酸。
  2. 如权利要求1所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异；
    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:362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；
    或者，所述核苷酸序列I与SEQ ID NO:481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:482所示的核苷酸序列之间不多于1个核苷酸差异。
  3. 如权利要求1或2所述的siRNA，其中，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间的核苷酸差异包括Z 4位置处的差异，且Z 4选自U、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U、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U、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U、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、U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U、C或G；
    或者，所述核苷酸序列II与SEQ ID NO:482所示的核苷酸序列之间的核苷酸差异包括Z 36位置处的差异，且Z 36选自A、C或G。
  4. 如权利要求1-3中任一项所述的siRNA，其中Z 3是与Z 4互补的核苷酸；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；或者，Z 35是与Z 36互补的核苷酸。
  5. 如权利要求1-4中任一项所述的siRNA，其中，所述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'-GGGUAUUCUUUCAAGCAAZ 3-3'(SEQ ID NO:3)；
    5'-Z 4UUGCUUGAAAGAAUACCC-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'-GGCAUAAACUAUAACAGCZ 7-3'(SEQ ID NO:63)；
    5'-Z 8GCUGUUAUAGUUUAUGCC-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'-GCUCAAGAAUGCCAAGAAZ 11-3'(SEQ ID NO:123)；
    5'-Z 12UUCUUGGCAUUCUUGAGC-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'-GCAACAAAGACAUUUAUGZ 15-3'(SEQ ID NO:183)；
    5'-Z 16CAUAAAUGUCUUUGUUGC-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'-GAAUCUCAAAGAAAUCUUZ 19-3'(SEQ ID NO:243)；
    5'-Z 20AAGAUUUCUUUGAGAUUC-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'-GUACGUGGACUGGAUUCUZ 23-3'(SEQ ID NO:303)；
    5'-Z 24AGAAUCCAGUCCACGUAC-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'-AUUUCUGGGUAUUCUUUCZ 27-3'(SEQ ID NO:363)；
    5'-Z 28GAAAGAAUACCCAGAAAU-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'-CAUGAAGGGCAUAAACUAZ 31-3'(SEQ ID NO:423)；
    5'-Z 32UAGUUUAUGCCCUUCAUG-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；
    或者，所述核苷酸序列I是SEQ ID NO:483所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:484所示的核苷酸序列：
    5'-GGAUUCUGGAGAAAACUCZ 35-3'(SEQ ID NO:483)；
    5'-Z 36GAGUUUUCUCCAGAAUCC-3'(SEQ ID NO:484)，
    其中，Z 35选自A、U、G或C，Z 36是与Z 35互补的核苷酸。
  6. [根据细则91更正 23.06.2020]　
    如权利要求5所述的siRNA，其中，Z 3为U，Z 4为A；或者Z 7为U，Z 8为A；或者Z 11为A，Z 12为U；或者Z 15为U，Z 16为A；或者Z 19为U，Z 20为A；或者Z 23为G，Z 24为C；或者Z 27为A，Z 28为U；或者Z 31为U，Z 32为A；或者Z 35为A，Z 36为U。
  7. [根据细则91更正 23.06.2020]　
    如权利要求1-6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。
  8. [根据细则91更正 23.06.2020]　
    如权利要求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CU；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UCU；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UUCU；
    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AG；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AAG；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GAAG；
    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AGU；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GAGU；
    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UU；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CUU；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GCUU；
    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AA；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AAA；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CAAA；
    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GA；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CGA；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UCGA；
    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CG；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GCG；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AGCG；
    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GA；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AGA；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UAGA；
    或者，所述核苷酸序列I与SEQ ID NO:481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CU；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，按照 5'末端到3'末端的方向，核苷酸序列III的碱基组成为ACU；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GACU。
  9. [根据细则91更正 23.06.2020]　
    如权利要求1-8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。
  10. [根据细则91更正 23.06.2020]　
    如权利要求9所述的siRNA，其中，所述核苷酸序列V的长度为2个核苷酸。
  11. [根据细则91更正 23.06.2020]　
    如权利要求9或10所述的siRNA，其中，所述核苷酸序列V为连续的两个胸腺嘧啶脱氧核糖核苷酸或连续的两个尿嘧啶核糖核苷酸，或者所述核苷酸序列V与靶mRNA相应位置的核苷酸互补。
  12. [根据细则91更正 23.06.2020]　
    如权利要求1-11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'-GGGUAUUCUUUCAAGCAAZ 3-3'(SEQ ID NO:5)；
    5'-Z 4UUGCUUGAAAGAAUACCCAG-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'-CUGGGUAUUCUUUCAAGCAAZ 3-3'(SEQ ID NO:7)；
    5'-Z 4UUGCUUGAAAGAAUACCCAGAA-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'-GGCAUAAACUAUAACAGCZ 7-3'(SEQ ID NO:65)；
    5'-Z 8GCUGUUAUAGUUUAUGCCCU-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'-AGGGCAUAAACUAUAACAGCZ 7-3'(SEQ ID NO:67)；
    5'-Z 8GCUGUUAUAGUUUAUGCCCUUC-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'-GCUCAAGAAUGCCAAGAAZ 11-3'(SEQ ID NO:125)；
    5'-Z 12UUCUUGGCAUUCUUGAGCAC-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'-GUGCUCAAGAAUGCCAAGAAZ 11-3'(SEQ ID NO:127)；
    5'-Z 12UUCUUGGCAUUCUUGAGCACUC-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'-GCAACAAAGACAUUUAUGZ 15-3'(SEQ ID NO:185)；
    5'-Z 16CAUAAAUGUCUUUGUUGCAA-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'-UUGCAACAAAGACAUUUAUGZ 15-3'(SEQ ID NO:187)；
    5'-Z 16CAUAAAUGUCUUUGUUGCAAGC-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'-GAAUCUCAAAGAAAUCUUZ 19-3'(SEQ ID NO:245)；
    5'-Z 20AAGAUUUCUUUGAGAUUCUU-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'-AAGAAUCUCAAAGAAAUCUUZ 19-3'(SEQ ID NO:247)；
    5'-Z 20AAGAUUUCUUUGAGAUUCUUUG-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 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'-GUACGUGGACUGGAUUCUZ 23-3'(SEQ ID NO:305)；
    5'-Z 24AGAAUCCAGUCCACGUACUC-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'-GAGUACGUGGACUGGAUUCUZ 23-3'(SEQ ID NO:307)；
    5'-Z 24AGAAUCCAGUCCACGUACUCGA-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'-AUUUCUGGGUAUUCUUUCZ 27-3'(SEQ ID NO:365)；
    5'-Z 28GAAAGAAUACCCAGAAAUCG-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'-CGAUUUCUGGGUAUUCUUUCZ 27-3'(SEQ ID NO:367)；
    5'-Z 28GAAAGAAUACCCAGAAAUCGCU-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'-CAUGAAGGGCAUAAACUAZ 31-3'(SEQ ID NO:425)；
    5'-Z 32UAGUUUAUGCCCUUCAUGUC-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'-GACAUGAAGGGCAUAAACUAZ 31-3'(SEQ ID NO:427)；
    5'-Z 32UAGUUUAUGCCCUUCAUGUCUA-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；
    或者，所述siRNA的正义链含有如SEQ ID NO:485所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:486所示的核苷酸序列：
    5'-GGAUUCUGGAGAAAACUCZ 35-3'(SEQ ID NO:485)；
    5'-Z 36GAGUUUUCUCCAGAAUCCAG-3'(SEQ ID NO:486)，
    或者，所述siRNA的正义链含有如SEQ ID NO:487所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:488所示的核苷酸序列：
    5'-CUGGAUUCUGGAGAAAACUCZ 35-3'(SEQ ID NO:487)；
    5'-Z 36GAGUUUUCUCCAGAAUCCAGUC-3'(SEQ ID NO:488)，
    其中，所述Z 36是反义链5'末端的第一个核苷酸，Z 35选自A、U、G或C，并且Z 36是与Z 35互补的核苷酸。
  13. [根据细则91更正 23.06.2020]　
    如权利要求1-12中任一项所述的siRNA，其中，所述siRNA为siFXIa1、siFXIa2、siFXIb1、siFXIb2、siFXIc1、siFXIc2、siFXId1、siFXId2、siFXIe1、siFXIe2、siFXIf1、siFXIf2、siFXIg1、siFXIg2、siFXIh1、siFXIh2、siFXIi1和siFXIi2中的任意一种。
  14. [根据细则91更正 23.06.2020]　
    如权利要求1-13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。
  15. [根据细则91更正 23.06.2020]　
    如权利要求1-14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。
  16. [根据细则91更正 23.06.2020]　
    如权利要求15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。
  17. [根据细则91更正 23.06.2020]　
    如权利要求16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。
  18. [根据细则91更正 23.06.2020]　
    如权利要求15-17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。
  19. [根据细则91更正 23.06.2020]　
    如权利要求18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。
  20. [根据细则91更正 23.06.2020]　
    如权利要求15-19中任一项所述的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。
  21. [根据细则91更正 23.06.2020]　
    如权利要求17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。
  22. [根据细则91更正 23.06.2020]　
    如权利要求1-21中任一项所述的siRNA，其中，所述siRNA为siFXIa1-M1、siFXIa1-M2、siFXIa1-M3、siFXIa2-M1、siFXIa2-M2、siFXIa2-M3、siFXIb1-M1、siFXIb1-M2、siFXIb1-M3、siFXIb2-M1、siFXIb2-M2、siFXIb2-M3、siFXIc1-M1、siFXIc1-M2、siFXIc1-M3、siFXIc2-M1、siFXIc2-M2、siFXIc2-M3、siFXId1-M1、siFXId1-M2、siFXId1-M3、siFXId2-M1、siFXId2-M2、siFXId2-M3、siFXIe1-M1、siFXIe1-M2、siFXIe1-M3、siFXIe2-M1、siFXIe2-M2、siFXIe2-M3、siFXIf1-M1、siFXIf1-M2、siFXIf1-M3、siFXIf2-M1、siFXIf2-M2、siFXIf2-M3、siFXIg1-M1、siFXIg1-M2、siFXIg1-M3、siFXIg2-M1、siFXIg2-M2、siFXIg2-M3、siFXIh1-M1、siFXIh1-M2、siFXIh1-M3、siFXIh2-M1、siFXIh2-M2、siFXIh2-M3、siFXIi1-M1、siFXIi1-M2、siFXIi1-M3、siFXIi2-M1、siFXIi2-M2和siFXIi2-M3中的任意一种。
  23. [根据细则91更正 23.06.2020]　
    如权利要求14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。
  24. [根据细则91更正 23.06.2020]　
    如权利要求14或23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 PCTCN2020091484-appb-100001
  25. [根据细则91更正 23.06.2020]　
    如权利要求23或24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。
  26. [根据细则91更正 23.06.2020]　
    如权利要求1-25中任一项所述的siRNA，其中，所述siRNA为siFXIa1-M1S、siFXIa1-M2S、siFXIa1-M3S、siFXIa2-M1S、siFXIa2-M2S、siFXIa2-M3S、siFXIa1-M1S1、siFXIa1-M2S1、siFXIa1-M3S1、siFXIa2-M1S1、siFXIa2-M2S1、siFXIa2-M3S1、siFXIb1-M1S、siFXIb1-M2S、siFXIb1-M3S、siFXIb2-M1S、siFXIb2-M2S、siFXIb2-M3S、siFXIc1-M1S、siFXIc1-M2S、siFXIc1-M3S、siFXIc2-M1S、siFXIc2-M2S、siFXIc2-M3S、siFXId1-M1S、siFXId1-M2S、siFXId1-M3S、siFXId2-M1S、siFXId2-M2S、siFXId2-M3S、siFXIe1-M1S、siFXIe1-M2S、siFXIe1-M3S、siFXIe2-M1S、siFXIe2-M2S、siFXIe2-M3S、siFXIf1-M1S、siFXIf1-M2S、siFXIf1-M3S、siFXIf2-M1S、siFXIf2-M2S、siFXIf2-M3S、siFXIg1-M1S、siFXIg1-M2S、siFXIg1-M3S、siFXIg2-M1S、siFXIg2-M2S、siFXIg2-M3S、siFXIh1-M1S、siFXIh1-M2S、siFXIh1-M3S、siFXIh2-M1S、siFXIh2-M2S、siFXIh2-M3S、siFXIi1-M1S、siFXIi1-M2S、siFXIi1-M3S、siFXIi2-M1S、siFXIi2-M2S和siFXIi2-M3S中的任意一种。
  27. [根据细则91更正 23.06.2020]　
    如权利要求1-26中任一项所述的siRNA，其中，所述siRNA为siFXIa1-M1P1、siFXIa1-M2P1、siFXIa1-M3P1、siFXIa2-M1P1、siFXIa2-M2P1、siFXIa2-M3P1、siFXIa1-M1SP1、siFXIa1-M2SP1、siFXIa1-M3SP1、siFXIa2-M1SP1、siFXIa2-M2SP1、siFXIa2-M3SP1、siFXIb1-M1P1、siFXIb1-M2P1、siFXIb1-M3P1、siFXIb2-M1P1、siFXIb2-M2P1、siFXIb2-M3P1、siFXIb1-M1SP1、siFXIb1-M2SP1、siFXIb1-M3SP1、siFXIb2-M1SP1、siFXIb2-M2SP1、siFXIb2-M3SP1、siFXIc1-M1P1、siFXIc1-M2P1、siFXIc1-M3P1、siFXIc2-M1P1、siFXIc2-M2P1、siFXIc2-M3P1、siFXIc1-M1SP1、siFXIc1-M2SP1、siFXIc1-M3SP1、siFXIc2-M1SP1、siFXIc2-M2SP1、siFXIc2-M3SP1、siFXId1-M1P1、siFXId1-M2P1、siFXId1-M3P1、siFXId2-M1P1、siFXId2-M2P1、siFXId2-M3P1、siFXId1-M1SP1、siFXId1-M2SP1、siFXId1-M3SP1、siFXId2-M1SP1、siFXId2-M2SP1、siFXId2-M3SP1、siFXIe1-M1P1、siFXIe1-M2P1、siFXIe1-M3P1、siFXIe2-M1P1、siFXIe2-M2P1、siFXIe2-M3P1、siFXIe1-M1SP1、siFXIe1-M2SP1、siFXIe1-M3SP1、siFXIe2-M1SP1、siFXIe2-M2SP1、siFXIe2-M3SP1、siFXIf1-M1P1、siFXIf1-M2P1、siFXIf1-M3P1、siFXIf2-M1P1、siFXIf2-M2P1、siFXIf2-M3P1、siFXIf1-M1SP1、siFXIf1-M2SP1、siFXIf1-M3SP1、siFXIf2-M1SP1、siFXIf2-M2SP1、siFXIf2-M3SP1、siFXIg1-M1P1、siFXIg1-M2P1、siFXIg1-M3P1、siFXIg2-M1P1、siFXIg2-M2P1、siFXIg2-M3P1、siFXIg1-M1SP1、siFXIg1-M2SP1、siFXIg1-M3SP1、siFXIg2-M1SP1、siFXIg2-M2SP1、siFXIg2-M3SP1、siFXIh1-M1P1、siFXIh1-M2P1、siFXIh1-M3P1、siFXIh2-M1P1、siFXIh2-M2P1、siFXIh2-M3P1、siFXIh1-M1SP1、siFXIh1-M2SP1、siFXIh1-M3SP1、siFXIh2-M1SP1、siFXIh2-M2SP1、siFXIh2-M3SP1、siFXIi1-M1P1、siFXIi1-M2P1、siFXIi1-M3P1、siFXIi2-M1P1、siFXIi2-M2P1、siFXIi2-M3P1、siFXIi1-M1SP1、siFXIi1-M2SP1、siFXIi1-M3SP1、siFXIi2-M1SP1、siFXIi2-M2SP1和siFXIi2-M3SP1中的任意一种。
  28. [根据细则91更正 23.06.2020]　
    一种药物组合物，其特征在于，该药物组合物含有权利要求1-27中任一项所述的siRNA和药学上可接受的载体。
  29. [根据细则91更正 23.06.2020]　
    如权利要求28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)。
  30. [根据细则91更正 23.06.2020]　
    如权利要求29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)。
  31. [根据细则91更正 23.06.2020]　
    如权利要求28-30中任一项所述的药物组合物，其中，所述药学上可接受的载体含有有机胺、辅助脂质和聚乙二醇化脂质；其中，所述有机胺为如式(201)所示的化合物和/或其药学上可接受的盐：
    Figure PCTCN2020091484-appb-100002
    其中：
    每个X 101和X 102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Y 101和Z 101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO 2；
    每个R 101、R 102、R 103、R 104、R 105、R 106和R 107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
    x是1-10的整数；
    n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R 102是氢；
    并且，如果n或m中的至少一个是2，那么R 103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
    Figure PCTCN2020091484-appb-100003
    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表示式(201)中的氮原子。
  32. [根据细则91更正 23.06.2020]　
    如权利要求31所述的药物组合物，其中，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 PCTCN2020091484-appb-100004
    所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
    所述聚乙二醇化脂质为1,2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
  33. [根据细则91更正 23.06.2020]　
    如权利要求31或32所述的药物组合物，其中，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)。
  34. [根据细则91更正 23.06.2020]　
    如权利要求33所述的药物组合物，其中，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(50-70):(20-40):(3-20)。
  35. [根据细则91更正 23.06.2020]　
    一种siRNA缀合物，所述siRNA缀合物含有权利要求1-27中任一项所述的siRNA以及缀合连接至该siRNA的缀合基团。
  36. [根据细则91更正 23.06.2020]　
    如权利要求35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。
  37. [根据细则91更正 23.06.2020]　
    如权利要求36所述的siRNA缀合物，其中，所述接头具有如式(301)所示的结构：
    Figure PCTCN2020091484-appb-100005
    其中，k为1-3的整数；
    L A为具有如式(302)所示结构的包含酰胺键的链状部分，每个所述L A在其两端分别与一个所述靶向基团和所述L C部分通过醚键相连接：
    Figure PCTCN2020091484-appb-100006
    L B为具有如式(303)所示结构的包含N-酰基吡咯烷的链状部分，所述链状部分在其一端具有羰基并与所述L C部分通过酰胺键相连接，在另一端具有氧原子并与所述siRNA通过磷酸酯键相连接：
    Figure PCTCN2020091484-appb-100007
    L C为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的2-4价连接基团，所述L C经由氧原子与各个所述L A部分通过醚键相连接，并且经由氮原子与所述L B部分通过酰胺键相连接。
  38. [根据细则91更正 23.06.2020]　
    如权利要求35-37中任一项所述的siRNA缀合物，其中，所述siRNA缀合物具有如式(305)所示的结构：
    Figure PCTCN2020091484-appb-100008
    其中，双螺旋结构表示所述siRNA。
  39. [根据细则91更正 23.06.2020]　
    如权利要求38所述的siRNA缀合物，其中，所述接头具有式(306)所示的结构：
    Figure PCTCN2020091484-appb-100009
    其中，l为0-3的整数；
    *表示所述接头上通过醚键与所述靶向基团连接的位点；
    #表示所述接头上通过磷酸酯键与所述siRNA连接的位点。
  40. [根据细则91更正 23.06.2020]　
    如权利要求35、36和39中任一项所述的siRNA缀合物，其中，所述siRNA缀合物具有如式(307)所示的结构：
    Figure PCTCN2020091484-appb-100010
    其中，双螺旋结构表示所述siRNA。
  41. [根据细则91更正 23.06.2020]　
    如权利要求36-40中任一项所述的siRNA缀合物，其中，所述接头连接至所述siRNA的正义链3'末端。
  42. [根据细则91更正 23.06.2020]　
    如权利要求35所述的siRNA缀合物，其中，所述缀合物具有式(308)所示的结构：
    Figure PCTCN2020091484-appb-100011
    其中，
    n1为选自1-3的整数，n3为选自0-4的整数；
    每个m1、m2和m3各自独立地为选自2-10的整数；
    每个R 10、R 11、R 12、R 13、R 14和R 15各自独立地为H，或选自于由以下基团所组成的组：C 1-C 10烷基、C 1-C 10卤代烷基以及C 1-C 10烷氧基；
    R 3为式A59所示结构的基团：
    Figure PCTCN2020091484-appb-100012
    其中，E 1为OH、SH或BH 2，Nu为权利要求1-26中任一项所述的siRNA；
    R 2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中R 2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2、-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
    每个L 1独立地是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，L 1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2，-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
    Figure PCTCN2020091484-appb-100013
    表示基团连接共价部分的位点；
    M 1表示靶向基团。
  43. [根据细则91更正 23.06.2020]　
    如权利要求42所述的siRNA缀合物，其中，每个L 1独立地选自于由基团A1-A26基团及其任意组合所组成的组：
    Figure PCTCN2020091484-appb-100014
    Figure PCTCN2020091484-appb-100015
    其中，每个j1独立地为1-20的整数；
    每个j2独立地为1-20的整数；
    每个R'独立地为C 1-C 10烷基；
    每个Ra选自于由A27-A45及其任意组合所组成的组：
    Figure PCTCN2020091484-appb-100016
    Figure PCTCN2020091484-appb-100017
    每个Rb独立地为C 1-C 10烷基；
    Figure PCTCN2020091484-appb-100018
    表示基团连接共价部分的位点。
  44. [根据细则91更正 23.06.2020]　
    如权利要求43所述的siRNA缀合物，其中，L 1选自于由基团A1、A4、A5、A6、A8、A10、A11、A13及其连接组合所组成的组。
  45. [根据细则91更正 23.06.2020]　
    如权利要求44所述的siRNA缀合物，其中，L 1为基团A1、A4、A8、A10和A11中至少2个的连接组合。
  46. [根据细则91更正 23.06.2020]　
    如权利要求45所述的siRNA缀合物，其中，L 1为基团A1、A8和A10中至少2个的连接组合。
  47. [根据细则91更正 23.06.2020]　
    如权利要求42-46中任一项所述的siRNA缀合物，其中，L 1的长度为3-25个原子。
  48. [根据细则91更正 23.06.2020]　
    如权利要求47所述的siRNA缀合物，其中，L 1的长度为4-15个原子。
  49. [根据细则91更正 23.06.2020]　
    如权利要求43-48中任一项所述的siRNA缀合物，其中，j1为2-10的整数，j2为2-10的整数，R'为C 1-C 4烷基，Ra为A27、A28、A29、A30和A31中的一种，Rb为C 1-C 5烷基。
  50. [根据细则91更正 23.06.2020]　
    如权利要求49所述的siRNA缀合物，其中，j1为3-5的整数，j2为3-5的整数，R'为甲基、乙基和异丙基中的一种，Ra为A27或A28，Rb为甲基、乙基、异丙基和丁基中的一种。
  51. [根据细则91更正 23.06.2020]　
    如权利要求42-50中任一项所述的siRNA缀合物，其中，n1为1-2的整数，n3为0-1的 整数，且n1+n3＝2-3。
  52. [根据细则91更正 23.06.2020]　
    如权利要求42-51中任一项所述的siRNA缀合物，其中，每个m1、m2和m3各自独立地为2-5的整数。
  53. [根据细则91更正 23.06.2020]　
    如权利要求42-52中任一项所述的siRNA缀合物，其中，m1＝m2＝m3。
  54. [根据细则91更正 23.06.2020]　
    如权利要求35-53中任一项所述的siRNA缀合物，其中，每个所述靶向基团独立地为与哺乳动物肝细胞表面的去唾液酸糖蛋白受体亲和的配体。
  55. [根据细则91更正 23.06.2020]　
    如权利要求54所述的siRNA缀合物，其中，每个所述靶向基团独立地为去唾液酸糖蛋白或糖。
  56. [根据细则91更正 23.06.2020]　
    如权利要求55所述的siRNA缀合物，其中，每个所述靶向基团独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳糖胺、N-异丁酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种。
  57. [根据细则91更正 23.06.2020]　
    如权利要求56所述的siRNA缀合物，其中，至少一个或每个所述靶向基团为半乳糖或N-乙酰半乳糖胺。
  58. [根据细则91更正 23.06.2020]　
    如权利要求42-57中任一项所述的siRNA缀合物，其中，R 10、R 11、R 12、R 13、R 14和R 15独立地为H、甲基或乙基。
  59. [根据细则91更正 23.06.2020]　
    如权利要求43-58中任一项所述的siRNA缀合物，其中，R 2上同时含有与含氮骨架上的N原子连接的连接位点和与R 3中的P原子连接的连接位点。
  60. [根据细则91更正 23.06.2020]　
    如权利要求42-59中任一项所述的siRNA缀合物，其中，R 2上所述与含氮骨架上的N原子连接的位点与N形成酰胺键，所述与R 3上的P原子连接的位点与P形成磷酸酯键。
  61. [根据细则91更正 23.06.2020]　
    如权利要求42-60中任一项所述的siRNA缀合物，其中，R 2选自B5、B6、B5'或B6'：
    Figure PCTCN2020091484-appb-100019
    其中，
    Figure PCTCN2020091484-appb-100020
    表示基团共价连接的位点，q 2为1-10的整数。
  62. [根据细则91更正 23.06.2020]　
    如权利要求61所述的siRNA缀合物，其中，q 2为1-5的整数。
  63. [根据细则91更正 23.06.2020]　
    如权利要求35、42-62中任一项所述的siRNA缀合物，其中，该缀合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：
    Figure PCTCN2020091484-appb-100021
    Figure PCTCN2020091484-appb-100022
    Figure PCTCN2020091484-appb-100023
    Figure PCTCN2020091484-appb-100024
    Figure PCTCN2020091484-appb-100025
    Figure PCTCN2020091484-appb-100026
  64. [根据细则91更正 23.06.2020]　
    如权利要求42-63中任一项所述的siRNA缀合物，其中，式A59中的P原子连接到siRNA正义链或反义链的端部，所述端部指所述正义链或反义链中从其一端起算的前4个核苷酸。
  65. [根据细则91更正 23.06.2020]　
    如权利要求64所述的siRNA缀合物，其中，式A59中的P原子连接到所述siRNA正义链或反义链的末端。
  66. [根据细则91更正 23.06.2020]　
    如权利要求65所述的siRNA缀合物，其中，式A59中的P原子连接到所述siRNA正义链的3'末端。
  67. [根据细则91更正 23.06.2020]　
    如权利要求42-66中任一项所述的siRNA缀合物，其中，式A59中的P原子通过磷酸二 酯键连接至所述siRNA中的核苷酸的2'位、3'位或5'位。
  68. [根据细则91更正 23.06.2020]　
    权利要求1-27中任一项所述的siRNA、权利要求27-33中任一项所述的药物组合物和/或权利要求35-67中任一项所述的siRNA缀合物在制备用于治疗和/或预防血栓性疾病和/或缺血性卒中的药物中的用途。
  69. [根据细则91更正 23.06.2020]　
    一种治疗和/或预防血栓性疾病和/或缺血性卒中的方法，其中，所述方法包括将有效量的权利要求1-27中任一项所述的siRNA、权利要求28-34中任一项所述的药物组合物和/或权利要求35-67中任一项所述的siRNA缀合物给予患有血栓性疾病和/或缺血性卒中的受试者。
  70. [根据细则91更正 23.06.2020]　
    一种抑制凝血因子XI基因表达的方法，该方法包括将有效量的权利要求1-27中任一项所述的siRNA、权利要求28-34中任一项所述的药物组合物和/或权利要求35-67中任一项所述的siRNA缀合物与所述细胞接触。
  71. [根据细则91更正 23.06.2020]　
    一种试剂盒，其中，该试剂盒含有权利要求1-27任一项所述的siRNA、权利要求28-34中任一项所述的药物组合物和/或权利要求35-67中任一项所述的siRNA缀合物。
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