WO2023213284A9 - 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 - Google Patents

一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 Download PDF

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WO2023213284A9
WO2023213284A9 PCT/CN2023/092178 CN2023092178W WO2023213284A9 WO 2023213284 A9 WO2023213284 A9 WO 2023213284A9 CN 2023092178 W CN2023092178 W CN 2023092178W WO 2023213284 A9 WO2023213284 A9 WO 2023213284A9
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nucleotide sequence
nucleotide
sirna
seq
nucleotides
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WO2023213284A1 (zh
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梁子才
张鸿雁
高山
郑书全
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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to a nucleic acid capable of inhibiting the expression of 17 ⁇ -hydroxysteroid dehydrogenase type 13 (HSD17B13 or 17beta-HSD13) gene and compositions and conjugates containing the nucleic acid.
  • the present disclosure also relates to methods of preparation and uses of these nucleic acids, compositions and conjugates.
  • 17 ⁇ -hydroxysteroid dehydrogenase type 13 (commonly known as HSD17B13 or 17beta-HSD13) is a member of the 17 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ -HSD) family.
  • 17 ⁇ -hydroxysteroid dehydrogenase type 13 is a protein located on the surface of lipid droplets in hepatocytes. Its function may be related to liver lipid metabolism and the production of transaminases. It is known that high expression levels are found in hepatocytes of the liver. , whereas lower levels can be detected in the ovaries, bone marrow, kidneys, brain, lungs, skeletal muscles, bladder, and testicles.
  • HSD17B13 levels are highly expressed in the livers of fatty liver or diabetic mice, and overexpression in the liver of C57BL/6 mice significantly increases lipogenesis and triglyceride (TG) content in the liver, leading to a fatty liver phenotype. .
  • HSD17B13 has been identified as a lipid droplet (LD)-related protein in patients with non-alcoholic fatty liver disease (NAFLD).
  • LD lipid droplet
  • NAFLD non-alcoholic steatohepatitis
  • HSD17B13 Knockdown of the gene expressing HSD17B13 attenuated oleic acid-induced LD formation in cultured hepatocytes. It has been reported that upregulation of HSD17B13 in the liver has been observed in patients with fatty liver disease, supporting the role of HSD17B13 in the pathogenesis of NAFLD.
  • siRNA with stabilizing modified nucleotides introduced into the sequence unexpectedly showed significantly higher HSD17B13 than siRNA without stabilizing modified nucleotides at the corresponding position. gene suppressor activity Therefore, the inventor made the following invention.
  • the present disclosure provides an siRNA comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I, the antisense strand comprising nucleotide sequence II, the nucleoside
  • Both the acid sequence I and the nucleotide sequence II are composed of 19 nucleotides, and each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified core.
  • the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to the first nucleotide sequence in the mRNA expressed by the HSD17B13 gene.
  • the stabilizing modified nucleotides refer to siRNAs in which the 2' hydroxyl group of the ribose sugar of the nucleotide is replaced by a stabilizing modification group, and the nucleotide at the corresponding position is an unmodified nucleotide.
  • the thermal stability of siRNA including the stabilizing modified nucleotide is increased, and the steric hindrance of the stabilizing modified group is greater than 2'-O-methyl.
  • the present disclosure also provides a pharmaceutical composition, which contains the siRNA provided by the present disclosure and a pharmaceutically acceptable carrier.
  • the disclosure further provides an siRNA conjugate, the siRNA conjugate comprising the siRNA provided by the disclosure and a conjugation group conjugated to the siRNA, the conjugation group comprising a linker and a pharmaceutically acceptable targeting group, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently connected, and each of the targeting groups is selected from the group capable of interacting with the cell surface.
  • Receptor-binding ligand comprising the siRNA provided by the disclosure and a conjugation group conjugated to the siRNA, the conjugation group comprising a linker and a pharmaceutically acceptable targeting group, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently connected, and each of the targeting groups is selected from the group capable of interacting with the cell surface.
  • Receptor-binding ligand the siRNA conjugate comprising the siRNA provided by the disclosure and a conjugation group conjugated to the siRNA, the conjugation group comprising a linker and a pharmaceutically acceptable
  • the present disclosure also provides the siRNA of the present disclosure and/or the pharmaceutical composition of the present disclosure and/or the siRNA conjugate of the present disclosure for use in the treatment and/or prevention of mRNA levels associated with HSD17B13 gene expression. Use in medicines for diseases or symptoms.
  • the present disclosure also provides a method of treating and/or preventing a disease or symptom associated with mRNA levels of HSD17B13 gene expression, the method comprising administering an siRNA of the present disclosure to a subject in need thereof, and /or a pharmaceutical composition of the present disclosure, and/or a siRNA conjugate of the present disclosure.
  • the present disclosure also provides a method for inhibiting HSD17B13 gene expression levels in cells, the method comprising administering an effective dose of the siRNA of the present disclosure, and/or the present disclosure
  • the pharmaceutical composition and/or siRNA conjugate of the present disclosure is contacted with the cell.
  • the present disclosure also provides a kit comprising the siRNA of the present disclosure, and/or the pharmaceutical composition of the present disclosure, and/or the siRNA conjugate of the present disclosure.
  • siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure has high inhibitory activity of HSD17B13 gene expression and/or low toxicity, and can effectively treat or prevent diseases or symptoms related to the mRNA level of HSD17B13 gene expression.
  • the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure exhibit excellent mRNA modulation activity of HSD17B13 gene expression.
  • the siRNA conjugate provided by the present disclosure has a high target sequence inhibitory activity in the psi-CHECK system.
  • the siRNA conjugate of the present disclosure has The inhibitory activity of the conjugate against the target sequence is greatly improved, and the IC50 value is only about 1/4 to 1/3 of the reference conjugate 1.
  • the siRNA conjugate provided by the present disclosure at a concentration of 50 nM or 5 nM has an inhibitory activity of HSD17B13 gene expression of mRNA of at least 91.30%, or even as high as 94.71%, and does not contain stabilization. Compared with the reference conjugates of modified nucleotides, the conjugates of the present disclosure all showed better HSD17B13 mRNA inhibitory activity.
  • the siRNA conjugate provided by the present disclosure can inhibit the mRNA expression of HSD17B13 gene by up to 92.4% at a concentration of 10 nM.
  • the siRNA conjugates provided by the present disclosure showed excellent HSD17B13 mRNA inhibitory activity at a concentration of 50nM or 5nM.
  • the inhibitory activity at the 50nM concentration was significantly higher than that at the 5nM concentration, and the highest concentration could reach 98%.
  • the inhibition rate of HSD17B13 mRNA of the siRNA conjugate provided by the present disclosure was 69.34%. Compared with the administration of a reference conjugate that did not contain stabilizing modified nucleotides, the conjugate of the present disclosure had an inhibitory rate of 69.34%. All showed more excellent HSD17B13 mRNA inhibitory activity.
  • siRNA conjugates of the disclosure comprising stabilizing modified nucleotides are administered. Finally, on the 8th day, the inhibition rate of HSD17B13 mRNA was 81.2%.
  • the inhibition rate of the siRNA conjugates provided by the present disclosure on off-target sequences in the psi-CHECK system was not higher than 50% within the test concentration range, showing that it was comparable to the reference conjugates that did not contain stabilizing modified nucleotides. Lower off-target effects than compounds.
  • the siRNA, pharmaceutical compositions and siRNA conjugates provided by the present disclosure can effectively inhibit the expression of the HSD17B13 gene, and at a dose that ensures safety, effectively treat and/or prevent mRNA level-related diseases expressed by the HSD17B13 gene. disease symptoms and has good application prospects.
  • Figure 1 is a bar graph showing the relative expression levels of HSD mRNA in monkey primary hepatocytes in vitro after transfection with 50 nM and 5 nM concentrations of siRNA of the present disclosure.
  • Figure 2 is a bar graph showing the relative expression levels of HSD mRNA in C57BL/6j mouse primary hepatocytes in vitro after transfection with 50 nM concentration of siRNA of the present disclosure.
  • Figure 3 is a graph showing the relative expression levels of HSD mRNA in mice after administration of 3 mg/kg of siRNA of the present disclosure.
  • Figure 4 is a bar graph showing the relative expression levels of HSD mRNA in monkey primary hepatocytes in vitro after transfection with 50 nM and 5 nM concentrations of siRNA of the present disclosure.
  • Figure 5 shows that after transfection of 10 nM and 1 nM concentrations of siRNA of the present disclosure, 24 hours Line graph of SEAP concentration in primary monkey hepatocytes at 1 and 72 hours in vitro.
  • Figure 6 is a graph showing the relative expression levels of HSD mRNA in mice after administration of 3 mg/kg of siRNA of the present disclosure and a reference conjugate.
  • HSD17B13 mRNA or "mRNA expressed by the HSD17B13 gene" refers to the mRNA having the sequence shown in the Genbank registration number NM_001136230.3, also referred to as HSD mRNA in this context, and the HSD17B13 gene refers to the transcription The above HSD17B13 mRNA gene.
  • the capital letters C, G, U, and A represent the base composition of nucleotides; the small letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy group.
  • P1 indicates that the nucleotide adjacent to the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • P1 is Represents 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 modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) Nucleotide, the letter combination Ps indicates that the nucleotide adjacent to the right side of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the nucleotide adjacent to the right side of the letter P is 5 '-Phosphonucleotide.
  • V-VP vinyl phosphate
  • E-VP vinyl phosphate
  • the letter combination Ps indicates that the nucleotide adjacent to the right side of the letter combination Ps is a phosphorothioate modified nucleotide
  • the capital letter P indicates that the nucleotide adjacent to the right side of the letter P is 5 '-Phosphonucleotide.
  • fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine
  • non-fluorinated modified nucleotide refers to Nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of a nucleotide with a non-fluorine group.
  • Nucleotide analogue refers to a nucleotide that can replace a nucleotide in a nucleic acid, but whose structure is different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymus Pyrimidine deoxyribonucleotide group. Such as isonucleotides, bridged nucleic acid (BNA) or acyclic nucleotides.
  • the "methoxy modified nucleotide” refers to the 2'- Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • the expressions "complementary” or “reverse complementary” are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are each associated with the other strand. The bases on them are paired in a complementary manner.
  • the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) always pairs with the pyrimidine base Pairs with cytosine (G).
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch in this field means that in double-stranded nucleic acids, the bases at corresponding positions do not pair in a complementary manner.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “ means that there is no more than one base mismatch between the two nucleotide sequences; “complete reverse complementarity” means that there is no base mismatch between the two nucleotide sequences.
  • nucleoside monomer refers to the method to be prepared according to Type and sequence of nucleotides in siRNA or siRNA conjugate, modified or unmodified nucleoside phosphoramidite monomers used in solid-phase phosphoramidite synthesis (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 this disclosure are all commercially available.
  • substituted or substituted groups such as substituted alkyl, substituted alkoxy, substituted amino, substituted aliphatic group, substituted A heteroaliphatic group, a substituted acyl group, a substituted aryl group or a substituted heteroaryl group.
  • substituted or substituted refers to a group formed by replacing the hydrogen atom in the group with one or more substituents.
  • substituted alkoxy refers to a group formed by replacing one or more hydrogen atoms in an alkoxy group with a substituent.
  • the substituent is selected from the group consisting of: C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 1 - C 10 haloalkyl, -OC 1 -C 10 alkyl, -OC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl -OH, -OC 1 -C 10 haloalkyl, -SC 1 -C 10 alkyl, -SC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl -SH, -SC 1 -C 10 haloalkyl, halogen substituent, -OH, -SH, -NH 2 , -
  • the substituent is C 1 -C 3 alkyl, C 6 -C 8 aryl, -OC 1 -C 3 alkyl, -OC 1 -C 3 alkylphenyl, halogen, - One of OH, -NH 2 , -CN or -NO 2 . It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible and/or inherently unstable. .
  • alkyl refers to straight and branched chains having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, such as from 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 particular number of carbons it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl” is meant to include n-butyl, sec-butyl , isobutyl and tert-butyl; “propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to the same residue as alkyl but with two points of attachment.
  • alkenyl refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond obtained by removing it from an adjacent carbon atom of the parent alkyl group. Obtained by removing a 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 base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1- En-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-diene-1 -yl, but-1,3-dien-2-yl, etc.
  • alkenyl groups have 2 to 20 carbon atoms, and 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 straight chain alkyl group having at least one carbon-carbon triple bond obtained by removing adjacent carbon atoms from the parent alkyl group. Obtained by removing two molecules of hydrogen.
  • Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.
  • alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl but with two points of attachment.
  • alkoxy refers to an alkyl group with the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentyloxy etc.
  • Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected through an oxygen bridge.
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removal of a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 6 to 18 carbon atoms of carbon, wherein at least one ring in the ring system is completely unsaturated, i.e., contains a cyclic structure according to Hückel's 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 the same residue as aryl but with two points of attachment.
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring free radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., contains 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 oxidized heteroatoms.
  • the heteroaryl group contains one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in the heteroaryl group are quaternized of nitrogen atoms. A heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl examples include, but are not limited to: azepantrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzobisoxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, 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 (benzodioxolyl), benzodioxinyl (benzodioxinyl), benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl , benzothien
  • halogen substituent or “halo” refers to fluoro, chlorine, bromo and iodo, and the term “halogen” includes fluorine, chlorine, bromine and iodine.
  • haloalkyl refers to an alkyl group as defined above in which the specified number of hydrogen atoms is replaced by one or more halogen atoms, up to the maximum allowed number.
  • haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.
  • hydroxyl protecting groups can be used in this disclosure.
  • protecting groups render a chemical functional group insensitive to specific reaction conditions and can be added to and removed from that functional group in the molecule without materially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, to Each of the above documents is incorporated herein by reference in its entirety.
  • the protecting group is stable under basic conditions but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups useful herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanth-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups useful herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4′′-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any species of poultry.
  • non-human primates e.g., rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any species of poultry.
  • treatment refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefit.
  • “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated.
  • therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, although the subject may still be suffering from the underlying disorder.
  • Prevention refers to a method of obtaining beneficial or desired results, including but not limited to preventive benefits.
  • a double-stranded siRNA, pharmaceutical composition, or siRNA conjugate may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of the disease, Even though it is possible that the diagnosis of the disease has not yet been made.
  • each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging linkage in the phosphodiester bond or phosphorothioate diester bond is
  • the oxygen atom or sulfur atom has a negative charge, and it can exist in the form of a hydroxyl or mercapto group.
  • the hydrogen ions in the hydroxyl or mercapto group can also be partially or completely replaced by cations.
  • the cation can be any cation, such as a metal cation, ammonium ion NH 4 + , or one of the organic ammonium cations.
  • the cation is selected from alkali metal ions, tertiary cations, etc.
  • the alkali metal cation 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.
  • the siRNA or siRNA conjugates of the present disclosure may exist at least partially in salt form.
  • the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond is at least partially combined with a sodium ion
  • the siRNA or siRNA conjugate of the present disclosure is expressed as a sodium salt or a partial sodium salt. form exists. Therefore, when referring to the siRNA or siRNA conjugate described in the present disclosure, including but not limited to the siRNA conjugate represented by any structural formula described in the present disclosure, it is intended to encompass the sodium salt of the siRNA or siRNA conjugate. or partial sodium salt form.
  • the present disclosure provides an siRNA with higher HSD17B13 gene inhibitory activity.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be described in detail here.
  • the siRNA of the present disclosure includes a sense strand and an antisense strand, the sense strand includes nucleotide sequence I, the antisense strand includes nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II Both consist of 19 nucleotides.
  • Each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide.
  • the nucleotide sequences I and The nucleotide sequence II is at least partially reverse complementary to the first nucleotide sequence, which is a 19-nucleotide nucleoside in the mRNA expressed by the HSD17B13 gene.
  • the stabilizing modified nucleoside Acid refers to a nucleotide in which the 2' hydroxyl group of the ribose sugar of the nucleotide is replaced by a stabilizing modifying group.
  • the thermal stability of the siRNA containing the stabilizing modified nucleotide is increased, and the steric hindrance of the stabilizing modified group is greater than 2'-O-methane. base.
  • the 3rd or 5th nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.
  • no more than 2 nucleotides from the 3rd to the 9th nucleotides in the nucleotide sequence II are the stabilizing modified nucleosides. acid.
  • the 3rd and/or 5th nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.
  • the third nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.
  • the fifth nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.
  • the nucleotides other than the 3rd to 9th nucleotides in the nucleotide sequence II are not stabilizing modified nucleotides. If at least one of the 3rd to 6th nucleotides in the nucleotide sequence II is a stabilizing modified nucleotide, the nucleotide sequence II includes a stabilizing modified nucleotide in addition to the 3rd to 9th nucleotides. , may significantly affect the ability to regulate the expression level of the siRNA's target sequence.
  • “increased thermal stability of siRNA” in the context of this disclosure refers to an increase in the thermal dissociation temperature (Tm) of the siRNA.
  • “increased thermal stability of double-stranded siRNA” refers to an increase in the Tm of the siRNA by at least 0.05°C, in some embodiments an increase of 0.1-6°C, and in some embodiments an increase in the Tm of 0.5-4 °C.
  • each stabilizing modification group independently has a structure represented by -XR, where X is O, NR', S or SiR' 2 ; R is C 2 -C 6 alkyl, One of substituted C 2 -C 6 alkyl, C 6 -C 8 aryl, and substituted C 6 -C 8 aryl, each R' is independently H, C 1 -C 6 alkyl, substituted One of C 1 -C 6 alkyl, C 6 -C 8 aryl, and substituted C 6 -C 8 aryl, so
  • the above-mentioned substituted C 2 -C 6 alkyl, substituted C 6 -C 8 aryl or substituted C 1 -C 6 alkyl refers to C 2 -C 6 alkyl, C 6 -C 8 aryl or C 1 - A group formed by replacing one or more hydrogen atoms in a C 6 alkyl group with a substituent group selected from one or more of the following substituents: C 1 -X
  • each of the stabilization modification groups Groups independently selected from 2'-O-methoxyethyl, 2'-O-allyl, 2'-allyl, 2'-O-2-N-methylamino-2-oxo One of ethyl, 2'-O-2-N,N-dimethylaminoethyl, 2'-O-3-aminopropyl and 2'-O-2,4-dinitrophenyl kind.
  • each said stabilizing modifying group is 2'-O-methoxyethyl.
  • the sense strand and the antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides,
  • the length ratio of the siRNA sense strand and the 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
  • the siRNA with stabilizing modified nucleotides of the present disclosure may be the first to seventh siRNA below, each of which is described separately below.
  • the siRNA of the present disclosure is a first siRNA.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and differ by no more than 3 nucleotides, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2
  • the nucleotide sequences are equal in length and differ by no more than 3 nucleotides:
  • Z a1 is A
  • Z a2 is U
  • the nucleotide sequence I includes a nucleotide Za3 whose position corresponds to Z a1
  • the nucleotide sequence II includes a nucleotide Za4 whose position corresponds to Z a2
  • the Z a4 is the The first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1.
  • position corresponding refers to the same position in the nucleotide sequence starting from the same end of the nucleotide sequence, for example, the first core at the 3' end of nucleotide sequence I
  • the nucleotide is the nucleotide at a position corresponding to the 1st nucleotide of SEQ ID NO:1.
  • the sense strand includes only nucleotide sequence I and 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: 1, and/or the nucleotide sequence II is different from SEQ ID NO:1. There is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:2.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 includes a difference at the Z a4 position, and Z a4 is selected from A, G or C.
  • the Z a3 is a nucleotide complementary to Z a4 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 is the difference at the Z a4 position, and Z a4 is selected from A, G or C.
  • each U or T in the nucleotide sequence of the siRNA of the present disclosure can be optionally replaced with each other. These nucleotide differences will not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effects of siRNA. These siRNAs containing nucleotide differences are also within the scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two nuclei There is a mismatch of no more than 3 bases between the nucleotide sequences; the substantial reverse complementarity refers to a mismatch of no more than 1 base between the two nucleotide sequences; the complete reverse complementarity Complementarity means there is no mismatch between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II are the same as the nucleotides at positions 1-17 of the first nucleotide sequence. Nucleotides are completely reverse complementary.
  • the nucleotide sequence II is completely opposite to the nucleotide sequence I.
  • the second nucleotide in the nucleotide sequence II is identical to the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.
  • There is a base mismatch between the 2nd nucleotide By including this base mismatch, the inhibitory activity of the siRNA of the present disclosure on the expression of the target gene can be further improved.
  • the second nucleotide in the nucleotide sequence II in the direction from the 5' end to the 3' end, is A; in the direction from the 3' end to the 5' end, the nucleoside The second nucleotide in acid sequence I is selected from A, G or C.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV, each nucleoside in nucleotide sequence III and nucleotide sequence IV
  • Each acid is independently a kind of non-fluorinated modified nucleotide and is not the stabilizing modified nucleotide
  • the length of the nucleotide sequence III and the nucleotide sequence IV is each 1-4 nucleosides.
  • nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary, and the core
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • the second nucleotide sequence refers to the mRNA expressed by the HSD17B13 gene and the aforementioned first section. A nucleotide sequence whose 5' end is adjacent and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and IV is each 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A, so The base of the second nucleotide sequence is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides acid, the base composition of the nucleotide sequence III is GU, the base composition of the nucleotide sequence IV is AC, the base composition of the second nucleotide sequence is GU; at this time, the sense The length ratio of the strand and the antisense strand is 21/21; alternatively, the lengths of the nucleotide sequences III and IV are both 3 nucleotides, the base composition of the nucleotide sequence III is GGU, and the The base composition of the nucleotide sequence IV is ACC, and
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, Therefore, the base composition of nucleotide sequence III is given, and the base composition of nucleotide base IV is determined.
  • the siRNA of the present disclosure is a second siRNA, wherein the nucleotide sequence 1 is equal in length to the nucleotide sequence shown in SEQ ID NO: 27, and differs by no more than 3 nucleotides. , and the length of the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO:28, and the difference is no more than 3 nucleotides:
  • Z b1 is U
  • Z b2 is A
  • the nucleotide sequence I includes the nucleotide Z b3 corresponding to Z b1
  • the nucleotide sequence II includes the nucleotide Z b2 corresponding to the position Z b4
  • the Z b4 is the first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 27.
  • the sense strand includes only nucleotide sequence I and 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:27, and/or the nucleotide sequence II is different from SEQ ID NO:27. There is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:28.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:28 includes a difference at the Z b4 position, and Z b4 is selected from U, G or C.
  • the nucleotide difference is a difference at position Z b4
  • Z b4 is selected from U, G, or C.
  • Z b3 is a nucleotide complementary to Z b4 .
  • each U or T in the nucleotide sequence of the siRNA of the present disclosure can be optionally replaced with each other. These nucleotide differences will not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effects of siRNA. These siRNAs containing nucleotide differences are also within the scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two nuclei There is no more than 3 base mismatches between the nucleotide sequences; the substantial reverse complementarity It means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II are the same as the nucleotides at positions 1-17 of the first nucleotide sequence. Nucleotides are completely reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or in the direction from the 5' end to the 3' end, the second one in the nucleotide sequence II There is a base mismatch between the nucleotide and the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.
  • the inhibitory activity of the siRNA of the present disclosure on the expression of the target gene can be further improved.
  • the second nucleotide in the nucleotide sequence II in the direction from the 5' end to the 3' end, is U; in the direction from the 3' end to the 5' end, the nucleoside The second nucleotide in acid sequence I is selected from U, G or C.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV, each nucleotide of nucleotide sequence III and nucleotide sequence IV Independently one of the non-fluorinated modified nucleotides and not the stabilizing modified nucleotide
  • the length of the nucleotide sequence III and the nucleotide sequence IV is each 1-4 nucleotides
  • the nucleotide sequence IV and the nucleotide sequence III are equal in length
  • the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary
  • the nucleoside The acid sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • the second nucleotide sequence refers to the mRNA expressed by the HSD17B13 gene and the aforementioned first section.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is The base is U, and the base of the second nucleotide sequence is A; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the nucleotide sequence III and the nucleotide The length of sequence IV is 2 nucleotides, the base composition of the nucleotide sequence III is CA, the base composition of the nucleotide sequence IV is UG, and the base composition of the second nucleotide sequence is The base composition is CA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the nucleotide sequence III and the nucleotide sequence The length of sequence IV is 3 nucleotides, the base composition of the nucleotide sequence III is UCA
  • the length ratio of the sense strand and the antisense strand is 22/22; or, the length of the nucleotide sequence III and the nucleotide sequence IV is both 4 nucleotides, and the The base composition of the nucleotide sequence III is CUCA, the base composition of the nucleotide sequence IV is UGAG, and the base composition of the second nucleotide sequence is CUCA; at this time, the sense strand and the antisense strand The length ratio of the chains is 23/23.
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide sequence IV is also determined. .
  • the siRNA of the present disclosure is a third siRNA.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:53 are equal in length, and differ by no more than 3 nucleotides, and the nucleotide sequence II and SEQ ID NO:54 The nucleotide sequences shown are equal in length and differ by no more than 3 nucleotides:
  • Z c1 is U
  • Z c2 is A
  • the nucleotide sequence I includes the nucleotide Z c3 whose position corresponds to Z c1
  • the nucleotide sequence II includes the nucleotide sequence corresponding to Z c2 Z c4
  • the Z c4 is the first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 53.
  • the sense strand includes only nucleotide sequence I and 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:53, and/or the nucleotide sequence II is different from SEQ ID NO:53. There is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:54.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:54 includes a difference at the Z c4 position, and Z c4 is selected from U, G, or C.
  • Z c3 is a nucleotide complementary to Z c4 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:54 is the difference at the Z c4 position, and Z c4 is selected from U, G or C.
  • each U or T in the nucleotide sequence of the siRNA of the present disclosure can be optionally replaced with each other. These nucleotide differences will not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effects of siRNA. These siRNAs containing nucleotide differences are also within the scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two nuclei There is a mismatch of no more than 3 bases between the nucleotide sequences; the substantial reverse complementarity refers to a mismatch of no more than 1 base between the two nucleotide sequences; the complete reverse complementarity Complementarity means there is no mismatch between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II are the same as the nucleotides at positions 1-17 of the first nucleotide sequence. Nucleotides are completely reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or in the direction from the 5' end to the 3' end, the second one in the nucleotide sequence II There is a base mismatch between the nucleotide and the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.
  • the inhibitory activity of the siRNA of the present disclosure on the expression of the target gene can be further improved, while the off-target effect is unexpectedly further reduced.
  • the second nucleotide in the nucleotide sequence II in the direction from the 5' end to the 3' end, is A; in the direction from the 3' end to the 5' end, the nucleoside The second nucleotide in acid sequence I is selected from A, G or C.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV, each nucleoside in nucleotide sequence III and nucleotide sequence IV
  • Each acid is independently a kind of non-fluorinated modified nucleotide and is not the stabilizing modified nucleotide
  • the length of the nucleotide sequence III and the nucleotide sequence IV is each 1-4 nucleosides.
  • nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary, and the core
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, which refers to the mRNA expressed by the HSD17B13 gene.
  • the length of the nucleotide sequences III and IV is each 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C, so The base of the second nucleotide sequence is G; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides acid, the base composition of the nucleotide sequence III is AG, the base composition of the nucleotide sequence IV is CU, and the base composition of the second nucleotide sequence is AG; at this time, the sense The length ratio of the strand and the antisense strand is 21/21; alternatively, the lengths of the nucleotide sequences III and IV are both 3 nucleotides, the base composition of the nucleotide sequence III is CAG, and the The base composition of the nucleotide sequence IV is CUG,
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide base IV is also determined. .
  • the siRNA of the present disclosure is a fourth siRNA.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:79 are equal in length and differ by no more than 3 nucleotides
  • the nucleotide sequence II and SEQ ID NO:80 The nucleotide sequences shown are equal in length and differ by no more than 3 nucleotides:
  • Z d1 is U
  • Z d2 is A
  • the nucleotide sequence I includes the nucleotide Z d3 whose position corresponds to Z d1
  • the nucleotide sequence II includes the nucleotide sequence corresponding to Z d2 Z d4
  • the Z d4 is the first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:79.
  • the sense strand includes only nucleotide sequence I and 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:79, and/or the nucleotide sequence II is different from SEQ ID NO:79.
  • the nucleotide sequences shown in ID NO:80 differ by no more than 1 nucleotide.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:80 includes a difference at the Z d4 position, and Z d4 is selected from U, G or C.
  • Z d3 is a nucleotide complementary to Z d4 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:80 is the difference at the Z d4 position, and Z d4 is selected from U, G or C.
  • each U or T in the nucleotide sequence of the siRNA of the present disclosure can be optionally replaced with each other. These nucleotide differences will not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effects of siRNA. These siRNAs containing nucleotide differences are also within the scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two nuclei There is a mismatch of no more than 3 bases between the nucleotide sequences; the substantial reverse complementarity refers to a mismatch of no more than 1 base between the two nucleotide sequences; the complete reverse complementarity Complementarity means there is no mismatch between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II are the same as the nucleotides at positions 1-17 of the first nucleotide sequence. Nucleotides are completely reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or in the direction from the 5' end to the 3' end, the second one in the nucleotide sequence II There is a base mismatch between the nucleotide and the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.
  • the inhibitory activity of the siRNA of the present disclosure on the expression of the target gene can be further improved.
  • the second nucleotide in the nucleotide sequence II is A; in the direction from the 3' end to the 5' end, the nucleoside The second nucleotide in acid sequence I is selected from A, G or C.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV, each nucleoside in nucleotide sequence III and nucleotide sequence IV
  • Each acid is independently a kind of non-fluorinated modified nucleotide and is not the stabilizing modified nucleotide
  • the length of the nucleotide sequence III and the nucleotide sequence IV is each 1-4 nucleosides.
  • nucleoside The acid sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary, and the nucleotide sequence III is connected at The 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II. Moreover, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • the second nucleotide sequence refers to the mRNA expressed by the HSD17B13 gene and the aforementioned first section. A nucleotide sequence whose 5' end is adjacent and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequences III and IV is each 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C, so The base of the second nucleotide sequence is G; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides acid, the base composition of the nucleotide sequence III is AG, the base composition of the nucleotide sequence IV is CU, and the base composition of the second nucleotide sequence is AG; at this time, the sense The length ratio of the strand and the antisense strand is 21/21; alternatively, the lengths of the nucleotide sequences III and IV are both 3 nucleotides, the base composition of the nucleotide sequence III is AAG, and the The base composition of the nucleotide sequence IV is CUU
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide base IV is also determined. .
  • the siRNA of the present disclosure is a fifth siRNA.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:105 are equal in length and differ by no more than 3 nucleotides
  • the nucleotide sequence II and SEQ ID NO:106 are equal in length and differ by no more than 3 nucleotides:
  • Z e1 is A
  • Z e2 is U
  • nucleotide sequence I includes a position corresponding to Z e1 Nucleotide Ze3
  • nucleotide sequence II includes nucleotide Ze4 whose position corresponds to Ze2
  • Ze4 is the first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 105.
  • the sense strand includes only nucleotide sequence I and 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: 105, and/or the nucleotide sequence II is different from SEQ ID NO:105. There is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:106.
  • the difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 106 is Z e4 , and Z e4 is selected from A, G, or C.
  • the Ze3 is a nucleotide complementary to Ze4 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 106 is the difference at the Z e4 position, and Z e4 is selected from A, G or C.
  • each U or T in the nucleotide sequence of the siRNA of the present disclosure can be optionally replaced with each other. These nucleotide differences will not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effects of siRNA. These siRNAs containing nucleotide differences are also within the scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two nuclei There is a mismatch of no more than 3 bases between the nucleotide sequences; the substantial reverse complementarity refers to a mismatch of no more than 1 base between the two nucleotide sequences; the complete reverse complementarity Complementarity means there is no mismatch between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II are the same as the nucleotides at positions 1-17 of the first nucleotide sequence. Nucleotides are completely reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or in the direction from the 5' end to the 3' end, the second one in the nucleotide sequence II There is a base mismatch between the nucleotide and the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.
  • the inhibitory activity of the siRNA of the present disclosure on the expression of the target gene can be further improved.
  • the second nucleotide in the nucleotide sequence II in the direction from the 5' end to the 3' end, is A;
  • the second nucleotide in the nucleotide sequence I is selected from A, G or C in the direction from the 3' end to the 5' end.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV, each nucleoside in nucleotide sequence III and nucleotide sequence IV
  • Each acid is independently a kind of non-fluorinated modified nucleotide and is not the stabilizing modified nucleotide
  • the length of the nucleotide sequence III and the nucleotide sequence IV is each 1-4 nucleosides.
  • nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary, and the core
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • the second nucleotide sequence refers to the mRNA expressed by the HSD17B13 gene and the aforementioned first section. A nucleotide sequence whose 5' end is adjacent and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and IV is each 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A, so The base of the second nucleotide sequence is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides acid, the base composition of the nucleotide sequence III is GU, the base composition of the nucleotide sequence IV is AC, the base composition of the second nucleotide sequence is GU; at this time, the sense The length ratio of the strand and the antisense strand is 21/21; alternatively, the lengths of the nucleotide sequences III and IV are both 3 nucleotides, the base composition of the nucleotide sequence III is AGU, and the The base composition of the nucleotide sequence IV is ACU, and
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide base IV is also determined. .
  • the sixth siRNA is the sixth siRNA
  • the siRNA of the present disclosure is a sixth siRNA.
  • the The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:131 are equal in length and differ by no more than 3 nucleotides
  • the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO:132.
  • the nucleotide sequences are equal in length and differ by no more than 3 nucleotides:
  • Z f1 is A
  • Z f2 is U
  • the nucleotide sequence I includes the nucleotide Z f3 corresponding to Z f1
  • the nucleotide sequence II includes the nucleotide Z f2 corresponding to the position Z f4
  • the Z f4 is the first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 131.
  • the sense strand includes only nucleotide sequence I and 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:131, and/or the nucleotide sequence II is different from SEQ ID NO:131.
  • the nucleotide sequences shown in ID NO:132 differ by no more than 1 nucleotide.
  • the difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 132 is Z the difference at position f4 , and Z f4 is selected from A, G, or C.
  • Zf3 is a nucleotide complementary to Zf4 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 132 is the difference at the Z f4 position, and Z f4 is selected from A, G or C.
  • each U or T in the nucleotide sequence of the siRNA of the present disclosure can be optionally replaced with each other. These nucleotide differences will not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effects of siRNA. These siRNAs containing nucleotide differences are also within the scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two nuclei There is a mismatch of no more than 3 bases between the nucleotide sequences; the substantial reverse complementarity refers to a mismatch of no more than 1 base between the two nucleotide sequences; the complete reverse complementarity Complementarity means there is no mismatch between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II are the same as the nucleotides at positions 1-17 of the first nucleotide sequence. Nucleotide complete Reverse complementation. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or in the direction from the 5' end to the 3' end, the second one in the nucleotide sequence II There is a base mismatch between the nucleotide and the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.
  • the inhibitory activity of the siRNA of the present disclosure on the expression of the target gene can be further improved.
  • the second nucleotide in the nucleotide sequence II is A; in the direction from the 3' end to the 5' end, the nucleoside The second nucleotide in acid sequence I is selected from A, G or C.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV, each nucleoside in nucleotide sequence III and nucleotide sequence IV
  • Each acid is independently a kind of non-fluorinated modified nucleotide and is not the stabilizing modified nucleotide
  • the length of the nucleotide sequence III and the nucleotide sequence IV is each 1-4 nucleosides.
  • nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary, and the core
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • the second nucleotide sequence refers to the mRNA expressed by the HSD17B13 gene and the aforementioned first section. A nucleotide sequence whose 5' end is adjacent and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequences III and IV is each 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U, so The base of the second nucleotide sequence is A; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides acid, the base composition of the nucleotide sequence III is UA, the base composition of the nucleotide sequence IV is UA, the base composition of the second nucleotide sequence is UA; at this time, the sense The length ratio of the nucleotide sequence III and the antisense strand is 21/21; alternatively, the lengths of the nucleotide sequences III and IV are both 3 nucleotides, the base composition of the nucleotide sequence III is AUA, and the The base composition of the nucleotide
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide base IV is also determined. .
  • the siRNA of the present disclosure is a seventh siRNA.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:157 are equal in length and differ by no more than 3 nucleotides, and the nucleotide sequence II and SEQ ID NO:158 The nucleotide sequences shown are equal in length and differ by no more than 3 nucleotides:
  • Z g1 is A
  • Z g2 is U
  • the nucleotide sequence I includes the nucleotide Z g3 whose position corresponds to Z g1
  • the nucleotide sequence II includes the nucleotide sequence corresponding to Z g2 Z g4
  • the Z g4 is the first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 157.
  • the sense strand includes only nucleotide sequence I and 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:157, and/or the nucleotide sequence II is different from SEQ ID NO:157. There is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:158.
  • the difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 158 is the difference at position g4 , and Zg4 is selected from A, G, or C.
  • the Zg3 is a nucleotide complementary to Zg4 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 158 is the difference at the Z g4 position, and Z g4 is selected from A, G or C.
  • each U or T in the nucleotide sequence of the siRNA of the present disclosure can be optionally replaced with each other. These nucleotide differences will not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effects of siRNA. These siRNAs containing nucleotide differences are also within the scope of the present disclosure.
  • said nucleotide sequence I and said nucleotide sequence II are substantially Reverse complementarity, substantially reverse complementarity or complete reverse complementarity; the substantially reverse complementarity refers to a mismatch of no more than 3 bases between the two nucleotide sequences; the substantially reverse complementarity Complementarity means that there is no more than 1 base mismatch between two nucleotide sequences; said complete reverse complementarity means that there is no mismatch between two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II are the same as the nucleotides at positions 1-17 of the first nucleotide sequence. Nucleotides are completely reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or in the direction from the 5' end to the 3' end, the second one in the nucleotide sequence II There is a base mismatch between the nucleotide and the second nucleotide in the nucleotide sequence I in the direction from the 3' end to the 5' end.
  • the inhibitory activity of the siRNA of the present disclosure on the expression of the target gene can be further improved.
  • the second nucleotide in the nucleotide sequence II in the direction from the 5' end to the 3' end, is U; in the direction from the 3' end to the 5' end, the nucleoside The second nucleotide in acid sequence I is selected from U, G or C.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV, each nucleoside in nucleotide sequence III and nucleotide sequence IV
  • Each acid is independently a kind of non-fluorinated modified nucleotide and is not the stabilizing modified nucleotide
  • the length of the nucleotide sequence III and the nucleotide sequence IV is each 1-4 nucleosides.
  • nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or completely reverse complementary, and the core
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • the second nucleotide sequence refers to the mRNA expressed by the HSD17B13 gene and the aforementioned first section. A nucleotide sequence whose 5' end is adjacent and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequences III and IV is each 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C, so The base of the second nucleotide sequence is G; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides acid, the base composition of the nucleotide sequence III is UG, the base composition of the nucleotide sequence IV is CA, and the second nucleotide sequence The base composition is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the lengths of the nucleotide sequences III and IV are both 3 nucleotides, and the nucleotide sequences
  • the base composition of sequence III is AUG, the base composition of the nucleotide sequence IV is CAU, and
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide base IV is also determined. .
  • nucleotide sequence V nucleotide modifications and modified sequences in siRNA are applicable to the above-mentioned siRNA of the present disclosure, such as the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, The fifth siRNA, the sixth siRNA and the seventh siRNA. That is, if not specified, the following description of siRNA should be regarded as referring to the seven siRNAs of the present disclosure.
  • siRNA also contains the nucleotide sequence V
  • the siRNA of the present disclosure such as the above-mentioned first siRNA, second siRNA, third siRNA, fourth siRNA, fifth siRNA, sixth siRNA or seventh siRNA, also contains the nucleotide sequence V”.
  • the sense strand and the antisense strand are different in length, and the antisense strand also contains a nucleotide sequence V, each nucleotide of the nucleotide sequence V is independently a non-fluorinated modified core. One of the nucleotides and not the stabilizing modified nucleotide.
  • the length of the nucleotide sequence V is 1 to 3 nucleotides and is connected to the 3' end of the antisense strand to form the antisense strand. 3' overhang.
  • the length ratio of the siRNA sense strand and the antisense strand provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26.
  • the length of the nucleotide sequence V is 2 nucleotides. Therefore, the length ratio of the siRNA sense strand and the antisense strand provided by the present disclosure can be 19/21, 21/23 or 23 /25.
  • each nucleotide in the nucleotide sequence V can be any nucleotide.
  • the nucleotide sequence V is two consecutive thymidine deoxygens. Ribonucleotide (dTdT), two consecutive uracil ribonucleotides (UU);
  • dTdT Ribonucleotide
  • UU uracil ribonucleotides
  • the target nucleotide sequence V is completely reverse complementary to the third nucleotide sequence, which refers to the mRNA expressed by the HSD17B13 gene.
  • the length ratio of the sense strand and the antisense strand of the present disclosure is 19/21 or 21/23.
  • the siRNA of the present disclosure has better mRNA silencing activity.
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 1, and the base composition of the third nucleotide sequence is is GU; the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:4:
  • Z a4 is the first nucleotide at the 5' end of the antisense strand
  • Z a3 is selected from A, U, G or C
  • Z a3 is a nucleotide complementary to Z a4 .
  • 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:
  • Z a4 is the first nucleotide at the 5' end of the antisense strand
  • Z a3 is selected from A, U, G or C
  • Z a3 is a nucleotide complementary to Z a4 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 29, and the base composition of the third nucleotide sequence is It's CA.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:29, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:30:
  • Z b4 is the first nucleotide at the 5' end of the antisense strand
  • Z b3 is selected from A, U, G or C
  • Z b4 is a nucleotide complementary to Z b3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:31, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:32:
  • Z b4 is the first nucleotide at the 5' end of the antisense strand
  • Z b3 is selected from A, U, G or C
  • Z b4 is a nucleotide complementary to Z b3 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 55, and the base composition of the third nucleotide sequence is is AG;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:55, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:56:
  • Z c4 is the first nucleotide at the 5' end of the antisense strand
  • Z c3 is selected from A, U, G or C
  • Z c4 is a nucleotide complementary to Z c3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:57
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:58:
  • Z c4 is the first nucleotide at the 5' end of the antisense strand
  • Z c3 is selected from A, U, G or C
  • Z c4 is a nucleotide complementary to Z c3 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 81, and the base composition of the third nucleotide sequence is is AG; the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:81, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:82:
  • Z d4 is the first nucleotide at the 5' end of the antisense strand
  • Z d3 is selected from A, U, G or C
  • Z d4 is a nucleotide complementary to Z d3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:83
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:84:
  • Z d4 is the first nucleotide at the 5' end of the antisense strand
  • Z d3 is selected from A, U, G or C
  • Z d4 is the nucleotide complementary to Z d3 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 107, and the base composition of the third nucleotide sequence is is GU; the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:107, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:108:
  • Z e4 is the first nucleotide at the 5' end of the antisense strand
  • Z e3 is selected from A, U, G or C
  • Z e4 is a nucleotide complementary to Z e3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:109
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:110:
  • Z e4 is the first nucleotide at the 5' end of the antisense strand
  • Z e3 is selected from A, U, G or C
  • Z e4 is a nucleotide complementary to Z e3 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 133, and the base composition of the third nucleotide sequence is is UA;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:133, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:134:
  • Z f4 is the first nucleotide at the 5' end of the antisense strand
  • Z f3 is selected from A, U, G or C
  • Z f4 is a nucleotide complementary to Z f3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:135, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:136:
  • Z f4 is the first nucleotide at the 5' end of the antisense strand
  • Z f3 is selected from A, U, G or C
  • Z f4 is a nucleotide complementary to Z f3 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 159, and the base of the third nucleotide sequence The base composition is UG; the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:159, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:160:
  • Z g4 is the first nucleotide at the 5' end of the antisense strand
  • Z g3 is selected from A, U, G or C
  • Z g4 is a nucleotide complementary to Z g3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:161
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:162:
  • Z g4 is the first nucleotide at the 5' end of the antisense strand
  • Z g3 is selected from A, U, G or C
  • Z g4 is a nucleotide complementary to Z g3 .
  • nucleotides in the siRNA of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleosides. These modifications on the acid and nucleotide groups will not cause the siRNA of the present disclosure to significantly weaken or lose its function of inhibiting HSD17B13 gene expression.
  • modified nucleotide is used to refer to a nucleotide or nucleotide analogue in which the 2' hydroxyl group of the ribosyl group of a nucleotide is replaced by another group, or a nucleotide analogue.
  • the bases on the nucleotides are nucleotides that are modified bases.
  • the modified nucleotides will not cause significant weakening or loss of the function of siRNA to inhibit gene expression.
  • modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 can be selected.
  • the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II if not the stabilizing modified nucleotide, It is a fluorinated modified nucleotide.
  • all nucleotides in the nucleotide sequence II are modified nucleotides; in the direction from the 5' end to the 3' end, the 2nd and 6th nucleotides in the nucleotide sequence II , 14, 16 nucleotides, if not the stabilizing modified nucleotides, are fluorinated modified nucleotides, and other nucleotides in the nucleotide sequence II are independently non-fluorinated.
  • a type of modified nucleotide In some embodiments, from 5' end to 3' end direction, the 7th to 9th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides.
  • all nucleotides in the nucleotide sequence I are modified nucleotides; in the direction from the 5' end to the 3' end, the 7-9th nucleotides in the nucleotide sequence I
  • Each nucleotide is a fluorinated modified nucleotide
  • the other nucleotides in the nucleotide sequence I are each independently a type of non-fluorinated modified nucleotide.
  • fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with a non-fluorinated group. A sort of.
  • Nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-deoxynucleoside One of the glycosides.
  • the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8).
  • the 2'-amino modified nucleotide (2'- NH2 ) is represented by formula (9).
  • the 2'-deoxynucleotide (DNA) is represented by formula (10):
  • base modifications include, but are not limited to, adding one or more methyl groups to a base.
  • thymine (T) is considered a type of base-modified uracil (U).
  • 5-methylcytosine is considered base modified cytosine (C) kind of.
  • Nucleotide analogues are those that can replace nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides Ribonucleotide group.
  • nucleotide analogs may be isonucleotides, bridged nucleic acid (BNA), or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation. The bridge is typically incorporated into the 2', 4'-position of the ribose sugar to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA, etc., wherein LNA is represented by formula (12), ENA is represented by formula (13), and cET BNA is represented by formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (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).
  • Isonucleotides are compounds formed by changing the positions of the bases on the ribose ring in nucleotides.
  • the isonucleotide can be a compound formed by moving the base from the 1'-position of the ribose ring to the 2'-position or 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 a non-fluorine group as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, above and below, the methoxy-modified nucleotide refers to the 2' of the ribosyl group -Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • each non-fluoro-modified nucleotide in the antisense strand is 2'-deoxynucleotides
  • each of the remaining non-fluoromodified nucleotides is methyl Oxygen-modified nucleotides
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide
  • the methoxy-modified nucleotide refers to the 2'-hydroxyl group of the ribose group being Nucleotides formed by methoxy substitution.
  • the siRNA comprising stabilizing modified nucleotides of the present disclosure is an siRNA having the following modifications: in the direction from the 5' end to the 3' end, in the sense strand, the nucleotide sequence I
  • the nucleotides at positions 7, 8, 9 or 5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleosides. acid; in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorinated.
  • nucleotides the nucleotide at position 3 or 5 in the antisense strand is a stabilizing modified nucleotide, and the nucleotide at position 18 is 2'-deoxynucleoside Acid- or methoxy-modified nucleotides, and the remaining nucleotides in the antisense strand are methoxy-modified nucleotides.
  • siRNA with the above modifications is not only low-cost, but also makes it difficult for ribonucleases in the blood to cleave nucleic acids, thereby increasing the stability of nucleic acids and making nucleic acids more resistant to nuclease hydrolysis.
  • the above modifications did not significantly reduce the inhibitory performance of siRNA, and siRNA with the above modifications also had low off-target effects.
  • the siRNA provided by the present disclosure is siHSDa1-M1, siHSDa1-M2, siHSDa2-M1, siHSDa2-M2, siHSDb1-M1, siHSDb1-M2, siHSDb2- listed in Table 1a-Ig below.
  • the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and antisense strand of siRNA provided by the present disclosure is a phosphate group having a modified group.
  • the phosphate group with the modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom; in some embodiments, the The phosphate group having a modified group is a phosphorothioate group having a structure shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain the high specificity and high affinity of base pairing.
  • the present disclosure provides siRNAs in which the phosphorothioate linkage is present at at least one of the group consisting of: the first and second cores at either end of the sense strand or the antisense strand. Between nucleotides; between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above.
  • the phosphorothioate group is linked to Linkages are present at all of the above positions except the 5' end of the sense strand.
  • phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand.
  • the phosphorothioate linkage is present at at least one of the following positions:
  • the siRNA of the present disclosure is siHSDa1-M1S, siHSDa1-M2S, siHSDa2-M1S, siHSDa2-M2S, siHSDb1-M1S, siHSDb1-M2S, siHSDb2-M1S, siHSDb2-M2S listed in Tables 1a-1g , HSDc1-M1S, siHSDc1-M2S, siHSDc2-M1S, siHSDc2-M2S, HSDd1-M1S, siHSDd1-M2S, siHSDd2-M1S, siHSDd2-M2S, siHSDe1-M1S, siHSDe1-M2S, siHSDe2-M1S, siHSDe2-M2S, siHSDf1 -M1S, siHSDf1-M2S, siHSDf2-M1S, siHSDf2-M2S, siHSDg1-M1S,
  • 5'-phosphate nucleotide can have the following structure:
  • R is selected from H, OH, methoxy, and fluorine
  • Base represents a nucleic acid base, selected from A, U, C, G, or T.
  • the 5'-phosphate nucleotide is a 5'-phosphate modified nucleotide represented by formula (2)
  • the 5'-phosphate analog modified nucleotide is a nucleotide containing vinyl phosphate ( 5'-(E)-vinylphosphonate, E-VP) modified nucleotides, as shown in formula (3), or phosphorothioate-modified nucleotides, as shown in formula (5).
  • the siRNA of the present disclosure is siHSDa1-M1P, siHSDa1-M2P, siHSDa2-M1P, siHSDa2-M2P, siHSDb1-M1P, siHSDb1-M2P, siHSDb2-M1P, siHSDb2- listed in Table 1a-1g below.
  • siRNAs provided by the present disclosure unexpectedly discovered that these siRNAs provided by the present disclosure not only have significantly enhanced plasma and lysosomal stability, but also retain high HSD mRNA inhibitory activity, and also have low off-target effects.
  • the siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (such as solid phase synthesis and liquid phase synthesis methods). Among them, solid-phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications, methods of preparing nucleoside monomers with corresponding modifications, and introducing modified nucleotide groups. siRNA methods are 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 can be a carrier commonly used in the field of siRNA delivery, such as but not limited to magnetic nanoparticles (such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon nanotubes ( carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), polylysine Acid (poly(L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), polyD Type or L-type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid)copolymer, PLGA), poly(aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate
  • the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1:( 1-500), in some embodiments, the above weight ratio is 1:(1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable auxiliary materials, which may be one or more of various preparations or compounds commonly used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg). Based on the required osmotic pressure, those skilled in the art can easily determine the content. In some embodiments, the dosage of the preparation made from the pharmaceutical composition during administration will be adjusted due to different administration methods.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation can be, but is not limited to, administered by subcutaneous, intramuscular or intravenous injection. It can also be administered to the lungs by spray, or to other organs and tissues (such as the liver) through the lungs. Or the pharmaceutical composition can be delivered orally. In some embodiments, the pharmaceutical composition is administered by subcutaneous injection.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipids.
  • an organic amine, auxiliary lipid and pegylated lipid can be selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (which is incorporated herein by reference in its entirety) or its One or more of pharmaceutically acceptable salts or derivatives, auxiliary lipids and pegylated lipids.
  • the organic amine can be a compound represented by formula (201) described in Chinese patent application 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, ring cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic groups, cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic groups Group, substituted or unsubstituted, branched or linear acyl group, substituted or unsubstituted, branched or linear aryl group, substituted or unsubstituted, branched or linear heterogeneous group Aryl;
  • x is an integer from 1 to 10;
  • n is an integer from 1 to 3
  • m is an integer from 0 to 20
  • R 103 and nitrogen in formula (201) form a structure 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 linear alkyl or alkenyl group, the alkyl 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 may be any of the following formulas (204) to (213):
  • g, e and f are each independently an integer from 1 to 6
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201)
  • Possible attachment points to the nitrogen atoms in where each H at any * position can be replaced to effect attachment to the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.
  • the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):
  • the auxiliary lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives
  • the pegylated lipid is 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, it can be (50-70):(20-40):(3-20).
  • the pharmaceutical composition particles formed from the siRNA of the present disclosure and the above-described amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the liposomes
  • 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.
  • the average diameter of the liposome particles is about 30, 40, 50, 60, 70 , 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.
  • the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent are formed.
  • the weight ratio (weight/weight ratio) of siRNA to 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, from about 1:5 to about 1:17, from about 1:5 to In the range of about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the siRNA of the present disclosure and all lipids
  • the mass-to-weight ratio is approximately 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.
  • each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used.
  • the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, and it is only necessary to replace the existing siRNA with the siRNA provided by the present disclosure; in some cases In an 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 obtained 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, polyethylene glycol 400 One or more, for example, it can be ethanol.
  • the siRNA provided by the present disclosure is dissolved in the buffer salt solution to obtain an siRNA aqueous solution.
  • the concentration of the buffer salt solution is 0.05-0.5M, for example, it can be 0.1-0.2M.
  • the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, it can be 5.0-5.2.
  • the amount of buffer salt solution is such that the concentration of siRNA does not exceed 0.6 mg. /mL, for example, it can be 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, it can be sodium acetate and/or potassium acetate.
  • the volume ratio of the lipid solution and the siRNA aqueous solution is 1:(2-5), for example, it can be 1:4.
  • the incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided by the present disclosure, the physical and chemical parameters of which are a pH value of 6.5-8 and an encapsulation rate of not less than 80%.
  • the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be a pH value of 7.2-7.6, an encapsulation rate of not less than 90%, and a particle size of 60- 100nm, the polydispersity 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 impurities are removed.
  • Various existing methods can be used to remove impurities, such as using a tangential flow system, a hollow fiber column, ultrafiltration at 100K Da, and the ultrafiltration exchange solution is phosphate buffer saline (PBS) with pH 7.4.
  • PBS phosphate buffer saline
  • the sterilization method can adopt various existing methods, for example, filtration sterilization can be performed on a 0.22 ⁇ m filter.
  • the present disclosure provides a siRNA conjugate comprising a siRNA provided by the present disclosure, and a conjugation group conjugated to the siRNA.
  • the conjugation group includes a linker and a pharmaceutically acceptable targeting group and/or a delivery aid group, and the siRNA, the linker, and the targeting group or the The delivery auxiliary group is sequentially covalently or non-covalently connected, each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor, and each delivery auxiliary group is selected from a ligand capable of increasing the conjugation of the siRNA.
  • the biocompatible group of the drug in the delivery target organ or tissue is sequentially covalently or non-covalently connected, each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor, and each delivery auxiliary group is selected from a ligand capable of increasing the conjugation of the siRNA.
  • conjugated means that two or more chemical moieties, each having a specific function, are covalently linked to each other; accordingly, a “conjugate” is Refers to a compound formed by covalent connections between various chemical parts.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugate should be understood as a collective name for multiple siRNA conjugates or a siRNA conjugate represented by a certain chemical formula, depending on the context.
  • conjugation molecule is understood to mean a specific compound that can be conjugated to siRNA via a reaction, ultimately forming the siRNA conjugate of the present disclosure.
  • the conjugation group includes at least one pharmaceutically acceptable targeting group, or further includes a linker, and the siRNA, the linker and the targeting group are connected in sequence .
  • the number of targeting groups is 1-6. In some embodiments, the number of targeting groups is 2-4.
  • the siRNA molecule may be non-covalently or covalently conjugated to the conjugation group, eg, may be covalently conjugated to the conjugation group.
  • the conjugation site of siRNA and the conjugation group can be at the 3' end or 5' end of the sense strand of siRNA, or at the antisense strand. The 5' end of the strand can also be within the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3' end of the sense strand of the siRNA.
  • the conjugation group can be attached to the phosphate group, the 2'-hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugation group can also be connected to the 3'-position hydroxyl group, in which case the nucleotides are connected via a 2'-5' phosphodiester bond.
  • the conjugation group is usually attached to the phosphate group of the nucleotide; when the conjugation group is attached to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose sugar ring or base.
  • the targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • the types of these linkers, targeting groups and the connection methods with siRNA can be found in the disclosure of WO2015006740A2, the entire content of which is incorporated herein by reference.
  • the siRNA and the conjugation group can be connected through acid-labile or reducible chemical bonds. In the acidic environment of cellular endosomes, these chemical bonds can be degraded, thereby leaving the siRNA in a free state.
  • the conjugation group can be connected to the sense strand of siRNA to minimize the impact of conjugation on siRNA activity.
  • the targeting group may be a ligand commonly used in the field of siRNA delivery, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.
  • At least one or each of said targeting groups is selected from ligands capable of binding to cell surface receptors expressing said HSD17B13 gene.
  • At least one or each of said targeting groups is selected from ligands capable of binding to mammalian liver parenchymal cell surface receptors (ASGPR).
  • each of the targeting groups is independently a ligand that has affinity for the asialoglycoprotein receptor on the surface of mammalian hepatocytes.
  • each of the targeting groups is independently a salivary Sour glycoprotein or sugar.
  • each of the targeting groups is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).
  • each of the targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylfuranose, L-xylfuranose, D -Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose , ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucofuranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D- Galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-galactofuranose, glucosamine, sia
  • k is an integer from 1 to 3;
  • L A has an amide bond-containing structure as shown in formula (302)
  • L B has an N-acylpyrrolidine-containing structure as shown in formula (303), containing a carbonyl group and an oxygen atom
  • L C is based on a hydroxymethyl group A linking group for aminomethane, dihydroxymethylaminomethane or trimethylolaminomethane;
  • n 302 , q 302 and p 302 are each independently an integer from 2 to 6.
  • n 302 , q 302 and p 302 are each independently an integer from 2 or 3;
  • n 303 is an integer from 4 to 16.
  • Select the location, n 303 is an integer from 8 to 12, Indicates the site at which a group is covalently attached.
  • each L A is connected to one of the targeting groups through an ether bond, and is connected through the formation of an ether bond between the oxygen atom of the hydroxyl group in the L C part and the L C part;
  • L B is connected through formula (303)
  • the carbonyl group in is connected to the nitrogen atom of the amino group in the L C part to form an amide bond, and is connected to the siRNA through the oxygen atom in formula (303) to form a phosphate bond or a phosphorothioate bond.
  • the siRNA conjugate provided by the present disclosure has a structure shown in formula (305):
  • Nu represents the siRNA provided by the present disclosure.
  • the linker in the siRNA conjugates of the present disclosure has the structure represented by formula (306):
  • n 306 is an integer from 0 to 3
  • each p 306 is independently an integer from 1 to 6,
  • the connecting group forms an ether bond with the targeting group through the oxygen atom marked by *;
  • the connecting group has at least one of the oxygen atoms marked by # One is connected to the siRNA by forming a phosphate bond or a phosphorothioate bond, and the remaining oxygen atoms marked with # are connected to hydrogen atoms to form a hydroxyl group, or to a C 1 -C 3 alkyl group to form a C 1 -C 3 alkyl group.
  • siRNA conjugates of the present disclosure have a structure shown in formula (307):
  • Nu represents the siRNA provided by the present disclosure.
  • the siRNA conjugate of the present disclosure has the structure 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;
  • R 10 , R 11 , R 12 , R 13 , R 14 or R 15 are each independently H, or selected from the group consisting of: C 1 -C 10 alkyl, C 1 -C 10 haloalkyl group and C 1 -C 10 alkoxy group;
  • R 3 has the structure shown in formula A59:
  • E 1 is OH, SH or BH 2 , and Nu represents the siRNA provided by the present disclosure
  • each M1 represents a targeting group, and its definition and selectable range are the same as above.
  • each M1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
  • L is defined as a linear alkylene group, it may not be a linear group or have a different name, such as an amine or an alkenyl group resulting from the above substitutions and/or substitutions.
  • the length of L is the number of atoms in the chain connecting the two points of attachment.
  • the ring resulting from the replacement of a carbon atom of the linear alkylene group (such as a heterocyclylene or heteroarylene group) is counted as one atom.
  • n 1 may be an integer from 1 to 3
  • n 3 may be an integer from 0 to 4.
  • n1 is an integer from 1 to 2
  • n3 is an integer from 0 to 1
  • n1+n3 2-3.
  • the spatial position between the multiple M1 ligands can be adapted to the M1 ligand and the liver surface asialoglycoprotein receptor.
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C
  • each of R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is independently selected from H, methyl and ethyl.
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are all H.
  • R 3 is a group with the structure shown in Formula A59, wherein E 1 is OH, SH or BH 2 . Based on the consideration of easy availability of preparation raw materials, in some embodiments, E 1 is OH or SH.
  • R2 is selected to achieve attachment to A59 from the N atom on the nitrogen-containing backbone.
  • nitrogen-containing skeleton refers to a chain structure in which carbon atoms and N atoms connected to R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are connected to each other. Therefore, R2 can be any linking group capable of linking the A59 group to the N atom on the nitrogen-containing backbone in a suitable manner.
  • the R 2 group when the siRNA conjugate of the present disclosure is prepared by a solid-phase synthesis process, the R 2 group needs to contain both a connection site to an N atom on the nitrogen-containing skeleton and a connection site to R 3 The connection site where the P atoms are connected.
  • the site connected to the N atom on the nitrogen-containing skeleton in R 2 forms an amide bond with the N atom, and the site with the N atom forms an amide bond.
  • the site where the P atom on R3 is connected forms a phosphate bond with the P atom.
  • R2 is 2-20 atoms in length, or 4-15 atoms in length.
  • R2 is B5, B6, B5' or B6':
  • the value range of q 2 may be an integer from 1 to 10. In some embodiments, q 2 is an integer from 1 to 5.
  • L1 is selected from a linked combination of one or more of the groups of formulas A1-A26.
  • L 1 is selected from the connection combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13; in some embodiments, L 1 is selected from A1, A4, A connection combination of at least 2 of A8, A10 and A11; in some embodiments, L 1 is selected from a connection combination of at least 2 of A1, A8 and A10.
  • L 1 can be 3-25 atoms in length, 3-20 atoms in length, 4-15 atoms in length, or 5-12 atoms in length. 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 atoms.
  • j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5.
  • R' is a C1-C4 alkyl group. 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 C1-C5 alkyl group. In some embodiments, Rb is one of methyl, ethyl, isopropyl and butyl.
  • j1, j2, R', Ra, Rb are each selected in formulas A1-A26 to achieve N connection between the M 1 ligand and the nitrogen-containing skeleton, and to enable the M 1 ligand to The spatial location is more suitable for M1 ligand binding to the liver surface asialoglycoprotein receptor.
  • siRNA conjugates of the present disclosure have formula (403), (404), (405), (406), (407), (408), (409), (410), (411) , the structure shown in (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):
  • Nu represents the siRNA of the present disclosure.
  • the P atom in Formula A59 can be connected to any possible position in the siRNA sequence.
  • the P atom in Formula A59 can be connected to any nucleotide in the sense strand or antisense strand of siRNA; in In some embodiments, the P atom in Formula A59 is connected to any nucleotide of the sense strand of siRNA.
  • the P atom in Formula A59 is connected to the end of the siRNA sense strand or the 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 connected to the end of the sense strand or antisense strand of siRNA; in some embodiments, the P atom in Formula A59 is connected to the 3' end of the sense strand of siRNA. In the case of being connected to the above position of the sense strand of siRNA, after the conjugate provided by the present disclosure enters the cell, upon unwinding, a separate siRNA antisense strand can be released to inhibit target gene expression through the RNAi mechanism.
  • the P atom in Formula A59 can be connected to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or the nucleotide on the base. In some embodiments, the P atom in Formula A59 can be connected to the 2', 3' 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 dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand, or the P atom in Formula A59 is substituted by replacing the 3' hydroxyl group in the siRNA sense strand.
  • the hydrogen in the 2'-hydroxyl group of a nucleotide is connected to the nucleotide, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl group of the 5' end nucleotide of the siRNA sense strand.
  • the inventors of the present disclosure have found that the siRNAs of the present disclosure and siRNA conjugates containing these siRNAs exhibit significantly improved stability in plasma and low off-target effects, while also exhibiting higher HSD17B13 silencing activity. Therefore, in some embodiments, the siRNA of the present disclosure may be one of the siRNAs shown in Tables 1a and 1b.
  • the capital letters C, G, U, and A represent the base composition of nucleotides;
  • the small letter m means that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the small letter f means The nucleotide adjacent to the left side of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter d indicates that the nucleotide adjacent to the right side of the letter d is a 2'-deoxynucleotide;
  • the underlined The capital letter S indicates that the nucleotide adjacent to the left side of the letter S is a stabilizing modified nucleotide;
  • the small letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphorothioate group;
  • P1 indicates that the The adjacent nucleotide on the right side of P1 is a 5'-phosphate nucleotide or
  • S represents a specific stabilizing modification such as moe , where the underlined letter combination moe indicates that a nucleotide adjacent to the left of the letter combination moe has 2'-O-methoxy. Ethyl-modified nucleotides.
  • P1 represents a specifically modified VP, Ps or P, wherein the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is vinyl phosphate (5'-(E)- vinylphosphonate, E-VP) modified nucleotides.
  • the letter combination Ps indicates that the nucleotide adjacent to the right side of the letter combination Ps is a phosphorothioate-modified nucleotide.
  • each U or T in the sequences listed in the above Tables 1a-1g can be arbitrarily replaced with each other, and will not have a significant impact on the activity or off-target effects of siRNA.
  • siRNA conjugates can be conjugated by methods that have been described in detail in the prior art. become. For example, methods for preparing various siRNA conjugates are described in detail in WO2015006740A2.
  • the siRNA conjugates of the present disclosure can also be obtained by means well known to those skilled in the art.
  • WO2014025805A1 describes the preparation method of the structure represented by formula (305), and Rajeev et al. describe the preparation method of the structure represented by formula (307) in ChemBioChem 2015, 16, 903-908.
  • Chinese patent application CN110959011A also discloses in detail the method for preparing the siRNA conjugate represented by formula (308). The contents of the above documents are incorporated into this article in their entirety by reference.
  • the siRNA conjugates of the present disclosure can also be combined with other pharmaceutically acceptable excipients.
  • the excipients can be one or more of various preparations or compounds commonly used in the art. For details, please refer to the above article on the present disclosure. Description of pharmaceutical compositions.
  • the present disclosure provides siRNAs of the present disclosure, and/or pharmaceutical compositions and/or siRNA conjugates for use in the treatment and/or prevention of diseases or symptoms associated with mRNA levels of HSD17B13 gene expression. Uses in medicines.
  • the present disclosure provides the use of the siRNA of the present disclosure, and/or pharmaceutical compositions and/or siRNA conjugates, in the preparation of a medicament for the treatment and/or prevention of chronic fibroinflammatory liver disease.
  • the present disclosure provides a method of treating and/or preventing a disease or symptom associated with mRNA levels of HSD17B13 gene expression, the method comprising administering to a subject in need thereof an siRNA of the present disclosure, and /or a pharmaceutical composition of the present disclosure and/or a siRNA conjugate of the present disclosure.
  • the disease or condition associated with mRNA levels of HSD17B13 gene expression is chronic fibroinflammatory liver disease.
  • the chronic fibroinflammatory liver disease is selected from the group consisting of: hepatitis, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, Alcoholic steatohepatitis (ASH), alcoholic fatty liver disease (ALD), hepatitis C (HCV)-related cirrhosis, drug-induced liver injury, and hepatocellular necrosis.
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • ASH Alcoholic steatohepatitis
  • ALD alcoholic fatty liver disease
  • HCV hepatitis C-related cirrhosis
  • drug-induced liver injury and hepatocellular necrosis.
  • the present disclosure also provides a method for inhibiting HSD17B13 gene level expression in cells, the method comprising combining an effective dose of the siRNA of the present disclosure, and/or a pharmaceutical composition and/or a siRNA conjugate with The cells are in contact.
  • siRNA By administering siRNA, pharmaceutical compositions and/or siRNA conjugates provided by the present disclosure to a subject in need, prevention and/or treatment can be achieved through the mechanism of regulating gene expression.
  • siRNA, pharmaceutical compositions and/or siRNA conjugates provided by the present disclosure can be used to prevent and/or treat the pathological conditions or diseases described herein, or can be used to prepare for the prevention and/or treatment of the pathological conditions or diseases described herein.
  • Drug can be used to prevent and/or treat the pathological conditions or diseases described herein, or can be used to prepare for the prevention and/or treatment of the pathological conditions or diseases described herein.
  • the term "administration/administration” refers to the administration of siRNA, pharmaceutical compositions, and/or siRNA conjugates by a method or pathway that at least partially localizes the siRNA, pharmaceutical composition, and/or siRNA conjugate to the desired site to produce the desired effect.
  • the composition and/or siRNA conjugate is placed into the subject.
  • 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, pharmaceutical compositions, and/or siRNA conjugates to a specific site compared to the entire body of the subject; whereas systemic administration results in the delivery of the siRNA, pharmaceutical composition, and/or siRNA conjugate. and/or siRNA conjugates are delivered to substantially the entire body of the subject. Given that the present disclosure is intended to provide a means of preventing and/or treating chronic fibroinflammatory liver disease, in some embodiments a mode of administration capable of delivering the drug to the liver is employed.
  • Administration to the subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, or transdermal. 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 daily, weekly, every two weeks, every three weeks, every month, every 2 months, every 3 months, every six months, or once or more per year.
  • the dosage of siRNA, pharmaceutical composition and/or siRNA conjugate described in the present disclosure can be a dosage conventional in the art, and the dosage can be determined according to various parameters, especially the age, weight and gender of the subject. .
  • Toxicity and efficacy can be measured in cell culture or experimental animals by standard pharmaceutical procedures, such as determining LD50 (the lethal dose that kills 50% of the population) and ED50 (the dose that causes 50% of the maximum response intensity in quantitative response, and qualitatively).
  • reaction refers to the dose that causes 50% of experimental subjects to have a positive reaction). Dosage ranges for humans can be derived based on data from cell culture assays and animal studies.
  • siRNA When administering siRNA, pharmaceutical compositions and/or siRNA conjugates of the present disclosure, for example, to male or female C57BL/6J or C3H/HeNCrlVr mice, 6-12 weeks old, weighing 18-25 g, with In the siRNA, pharmaceutical composition and/or siRNA conjugate
  • the amount of siRNA can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in In further embodiments, it is 0.05-20 mg/kg body weight, in still further embodiments, it is 0.1-15 mg/kg body weight, in still further embodiments, it is 0.1-10 mg/kg body weight.
  • the above amounts may be preferred when administering siRNA, pharmaceutical compositions and/or siRNA conjugates of the present disclosure.
  • siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure into cells, the purpose of inhibiting the expression of the HSD17B13 gene in cells can also be achieved through the mechanism of RNAi.
  • the method provided by the present disclosure is used to inhibit the expression of HSD17B13 gene in cells.
  • the dosage of siRNA in the provided siRNA, pharmaceutical composition and/or siRNA conjugate can be easily determined by those skilled in the art based on the desired effect.
  • the amount of siRNA in a provided siRNA conjugate is an amount sufficient to reduce expression of the target gene and result in 1 pM to 1 ⁇ M, or 0.01 nM to 100 nM, or 0.01 nM to 100 nM at the target cell surface. 0.05 nM to 50 nM or to an extracellular concentration of about 5 nM.
  • the amount required to achieve this local concentration will vary depending on various factors, including the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cells or tissue, whether the delivery is local or systemic, etc.
  • the concentration at the delivery site can be significantly higher than the concentration at the surface of the target cells or tissue.
  • the present disclosure provides a kit comprising an effective amount of the siRNA, pharmaceutical composition and/or siRNA conjugate provided by the present disclosure.
  • kits described herein can provide siRNA, pharmaceutical compositions, and/or siRNA conjugates in one container.
  • a kit described herein may include a container providing a pharmaceutically acceptable excipient.
  • the kit may also include other ingredients, such as stabilizers or preservatives.
  • the kits described herein can include at least one additional therapeutic agent in a container other than the container in which the siRNA, pharmaceutical compositions, and/or siRNA conjugates described herein are provided.
  • the kit may include a method for combining siRNA, pharmaceutical compositions and/or siRNA conjugates with pharmaceutically acceptable carriers and/or excipients or other ingredients (if any). Instructions for mixing.
  • the siRNA and pharmaceutically acceptable carriers and/or auxiliary materials and the pharmaceutical composition and/or siRNA conjugate, and/or pharmaceutically acceptable auxiliary materials can be provided in any form, For example, liquid form, dry form or lyophilized form.
  • the siRNA and pharmaceutically acceptable carriers and/or excipients and the pharmaceutical compositions and/or siRNA conjugates and optional pharmaceutically acceptable excipients are substantially pure and/or free of bacteria.
  • sterile water can be provided in the kits of the present disclosure.
  • the reagents and culture media used in the following examples are all commercial products.
  • the nucleic acid electrophoresis, real-time PCR and other operations used are all described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). method to proceed.
  • the conjugates 1-7 in the following Table 2 were prepared. The only difference is that the sense strand and antisense strand of siRNA contained in each siRNA conjugate are as shown in Table 2 respectively. As shown in; according to the nucleic acid sequences of the siRNAs numbered Conjugate 1 to Conjugate 7 in Table 2 below, the sense strand and antisense strand of siRNA were synthesized respectively.
  • Nu in formula (403) is a siRNA group having the siRNA sequence corresponding to conjugates 1-7 in Table 2 of the present disclosure, and the conjugation group is connected to the 3' terminal nucleotide of the sense strand of the siRNA group The 3' position of ribose. Comparative Preparation Examples 1-9 Synthesis of Reference siRNA Conjugates
  • Sense strand and antisense strand Use ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25°C)) to dilute each reference siRNA conjugate to a concentration of 0.2mg/mL (based on siRNA), and use liquid quality A mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier) was used for molecular weight detection. The measured values are consistent with the theoretical values, indicating that the synthesized reference conjugate 1, reference conjugate 2, reference conjugate 3, reference conjugate 4, reference conjugate 5, reference conjugate Substance 6, reference conjugate 7, reference conjugate 8 and reference conjugate NC respectively have target designed double-stranded nucleic acid sequences.
  • LC-MS Liquid Chromatography-Mass Spectrometry
  • each reference siRNA conjugate has the structure shown in formula (403), and the included siRNA has the reference conjugate 1, reference conjugate 2, reference conjugate 4, reference conjugate 2 in Table 2.
  • reference conjugate 1 is a siRNA conjugate that has substantially the same sequence composition as conjugate 1, but does not contain stabilizing modified nucleotides;
  • reference conjugate 2 is the same as conjugate 2.
  • Reference Conjugate 3 is a siRNA conjugate that has substantially the same sequence composition as Conjugate 3, but does not contain stabilizing modified nucleotides
  • Reference Conjugate 4 is a siRNA conjugate that has essentially the same sequence composition as Conjugate 4, but does not contain stabilizing modified nucleotides
  • Reference Conjugate 5 has essentially the same sequence composition as Conjugate 5.
  • reference conjugate 6 has essentially the same sequence composition as conjugate 6, but does not contain stabilizing modified nucleotides.
  • siRNA conjugate is a siRNA conjugate with substantially the same sequence composition as conjugate 7, but does not contain stabilizing modified nucleotides; reference conjugate 8 is and Conjugate 9 is compared to siRNA conjugates that have essentially the same sequence composition but do not contain stabilizing modified nucleotides; reference conjugate NC is a negative sequence with no significant homology to any known mRNA. Control siRNA conjugate.
  • the uppercase letters C, G, U, A, and T represent the base composition of nucleotides;
  • the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide;
  • the underlined letter combination moe indicates that the nucleotide adjacent to the left of the letter combination moe is ribose 2'-O -Nucleotide modified by methoxyethyl;
  • 5mC moe indicates a nucleotide whose base is 5-methylcytosine and the ribose sugar is modified by 2'-O-methoxyethyl;
  • the lowercase letter s indicates this letter
  • the two nucleotides to the left and right of s are connected by a phosphorothioate group;
  • conjugates 8-10 in Table 2 above were prepared. The only difference is that the sense strand and antisense strand of siRNA contained in each siRNA conjugate are as shown in Table 2 respectively. As shown in; according to the nucleic acid sequences of the siRNAs numbered Conjugate 8 to Conjugate 12 in Table 2 above, the sense strand and antisense strand of siRNA were synthesized respectively.
  • Each siRNA conjugate has a structure represented by formula (403), wherein the conjugation group is covalently connected to the 3' position of the ribose ring of the 3' terminal nucleotide of the siRNA sense strand, and the siRNA conjugate contains siRNA
  • the groups respectively have the siRNA sequences corresponding to conjugates 8-12 in Table 2.
  • Table 2A For specific molecular weight detection results, see Table 2A below: Table 2A Molecular weight detection results of siRNA conjugates
  • each of the above-prepared conjugate 11, conjugate 12, reference conjugate 10 and reference conjugate 11 was prepared into a 0.02 mg/mL solution using 1 ⁇ PBS buffer as a test product solution.
  • the double-stranded thermal dissociation temperature Tm was calculated from the first derivative of the temperature-absorbance curve according to the spectrophotometer instructions. The Tm value and ⁇ Tm value results are shown in Table 2B below:
  • ⁇ Tm value (conjugate to be tested) Tm (conjugate to be tested) - Tm (reference conjugate 11).
  • 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 construct a detection plasmid, co-transfect the detection plasmid and the conjugate to be tested into HEK293A cells, and reflect the target sequence of siRNA through the expression level of the dual-luciferase reporter gene inhibitory activity. Specific steps are as follows:
  • Detection plasmids were constructed using psi-CHECK TM -2 (Promega TM ) plasmid, and detection plasmids were constructed based on conjugate 1, conjugate 2, and conjugate 4, which contained target sequence 1 and target sequence 2 respectively. and target sequence 3, that is, the target sequence of siRNA conjugate 1 is target sequence 1, the target sequence of siRNA conjugate 2 is target sequence 2, and the target sequence of siRNA conjugate 4 is target sequence 3.
  • the target sequence is as follows:
  • the target sequence 1, target sequence 2 or target sequence 3 is homologous to a part of the HSD mRNA and is completely complementary to the sequence of the antisense strand in the detected siRNA conjugate. Therefore, the effect of each siRNA conjugate on the target sequence 1 is The inhibitory effect can reflect the ability of the detected siRNA conjugate to inhibit the expression of the target gene. Clone target sequence 1 and its complementary sequence into Xho I/Not I sites of psi-CHECK TM -2 plasmid.
  • H-DMEM complete medium HyClone company
  • FBS fetal bovine serum
  • RMBIO fetal bovine serum
  • penicillin-streptomycin HyClone company
  • HEK293A cells were seeded in a 96-well plate at 8 ⁇ 10 3 cells/well. When the cell growth density reached 70% after 16 hours, the complete medium in the culture wells was drained, and 80 ⁇ L opti-MEM medium (GIBCO Company) was added to each well. ) and continue culturing for 1.5 hours.
  • siRNA conjugates used were Conjugate 1, Conjugate 2, and Conjugate 4 prepared above, and Reference Conjugate 1, Reference Conjugate 2, and Reference Conjugate 4 respectively.
  • Each 1A1-1A11 solution contains 1 ⁇ L of siRNA working solution of the above 11 concentrations, 0.05 ⁇ L of detection plasmid working solution (containing 10 ng of detection plasmid) and 8.95 ⁇ L of Opti-MEM medium.
  • Each 1B solution contains 0.2 ⁇ L Lipofectamine TM 2000 and 9.8 ⁇ L Opti-MEM medium.
  • Each 1C solution contains 0.05 ⁇ L of detection plasmid working solution (containing 10 ng of detection plasmid) and 8.95 ⁇ L of Opti-MEM culture medium.
  • each siRNA conjugate In the culture wells, add 1X1-1X11 of the transfection complex of each siRNA conjugate, and mix evenly. The addition amount is 20 ⁇ L/well, and the final concentration of each siRNA conjugate is approximately 40nM, 10nM, and 2.5nM respectively.
  • the transfection complexes 1X1-1X11 were all transfected into three culture wells to obtain a co-transfection mixture containing siRNA conjugates, which was recorded as the test group.
  • transfection complex 1Xa For each siRNA conjugate, add transfection complex 1Xa to the other three culture wells at an amount of 20 ⁇ L/well to obtain a transfection mixture without siRNA conjugate, which is recorded as a blank control group.
  • H-DMEM complete medium containing 20% FBS was added to each well. Place the 96-well plate in a CO2 incubator and continue culturing for 24 hours at 37°C in an air atmosphere containing 5v/v% CO2 .
  • the luminescence ratio Ratio Ratio (test) or Ratio (control) of each test group or control group is the average of the Ratios of the three culture wells; take the luminescence of the control group Using the ratio as the benchmark, the luminescence ratio of each test group was normalized to obtain the ratio R of Ratio (test)/Ratio (control), which represents the relative expression level of the Renilla reporter gene, that is, the residual activity.
  • the inhibition rate of siRNA on the target sequence (1-R) ⁇ 100%.
  • the IC 50 value of the target sequence targeted by the siRNA to be tested is calculated.
  • the function is as follows,
  • Y is the ratio R, which is the relative residual activity of Renilla
  • X is the logarithm of the transfected 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 corresponding to when Y is halfway between bottom and top
  • HillSlope is the slope of the curve at X'.
  • the siRNA conjugates of the present disclosure have high target sequence inhibitory activity in the in vitro psi-CHECK system.
  • the IC 50 value of the reference conjugate 1 is the IC 50 of the conjugate 1
  • the IC 50 value of reference conjugate 2 was more than 5 times that of conjugate 2, and conjugate 4 also showed extremely low IC 50 value for target sequence inhibition.
  • CM Culture medium The Primary Cell Solution
  • CM Culture medium The Primary Cell Solution
  • the frozen monkey primary hepatocytes (The Primary Cell Solution) were thawed in thawing medium (The Primary Cell Solution Company) containing 10% fetal bovine serum (FBS, RMBIO Company) at 37°C for 2 minutes. , centrifuge at 700 rpm for 5 min, discard the supernatant, dissolve with seeding medium (CM seeding medium, The Primary Cell Solution Company) and count using a cell counter, then add seeding medium until the number of viable cells is 4 ⁇ 10 5 cells/well were cultured overnight at 37°C in an incubator containing 5% CO 2 /95% air.
  • CM seeding medium The Primary Cell Solution Company
  • CM Culture medium The Primary Cell Solution
  • CM Culture medium The Primary Cell Solution
  • Each 2A solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 6 working solution and 97 ⁇ l of cell maintenance medium.
  • Each 2B solution contains 3 ⁇ l of the above-mentioned siRNA Conjugate 7 working solution and 97 ⁇ l of cell maintenance medium.
  • Each 2C solution contains 3 ⁇ l of the above-mentioned siRNA reference conjugate 6 working solution and 97 ⁇ l of cell maintenance medium.
  • Each 2D solution contains 3 ⁇ l of the above-mentioned siRNA reference conjugate 7 working solution and 97 ⁇ l of cell maintenance medium.
  • Each 2E solution contains 2 ⁇ l Lipofectamine TM 2000 and 98 ⁇ l cell maintenance medium.
  • transfection complex 2X a of siRNA Conjugate 6 respectively, mix evenly, and add 200 ⁇ l/well to obtain a final concentration of siRNA Conjugate 6 (based on siRNA) of about 50 nM.
  • the transfection mixture of transfection complexes was designated as test group 1.
  • transfection complex 2X b of siRNA Conjugate 7 In the two culture wells, add the transfection complex 2X b of siRNA Conjugate 7 respectively, mix evenly, and add 200 ⁇ l/well to obtain a final concentration of siRNA Conjugate 7 (based on siRNA) of about 50 nM.
  • the transfection mixture of transfection complexes was designated as test group 2.
  • test group 4 In the two culture wells, add 2X d of transfection complex of siRNA reference conjugate 7 respectively, mix evenly, and add 200 ⁇ l/well to obtain the final concentration of reference siRNA conjugate 7 (calculated as siRNA) The transfection mixture of approximately 50 nM was designated as test group 4.
  • the 12-well plate containing the transfection mixture containing siRNA conjugate 6, conjugate 7, reference conjugate 6 and reference conjugate 7 was placed in an incubator containing 5% CO 2 /95% air. Continue culturing at 37°C for 24 hours.
  • RNA in the cells in each well was extracted using the UNIQ-10 column total RNA extraction kit (purchased from Sangon Company, Cat. No. B511361-0100) according to the method described in the instructions, and solutions containing total RNA were obtained.
  • RNA is reverse transcribed.
  • the conditions for reverse transcription are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 50°C for 50 minutes, then incubate at 85°C for 5 minutes, and finally incubate at 4°C for 30 seconds. After the reaction is completed, proceed to the reverse transcription reaction. Add 80 ⁇ l of DEPC water to the system to obtain 100 ⁇ L of cDNA-containing solution.
  • each reverse transcription reaction system For each reverse transcription reaction system, take 5 ⁇ l of the above cDNA-containing solution as a template, and use The reagents provided by the SYBR qPCR SuperMix Plus kit (purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B) are configured with a qPCR reaction system of 20 ⁇ l. Among them, the PCR primer sequences used to amplify the target gene HSD and the internal reference gene GAPDH are as shown in Table 4 As shown, the final concentration of each primer is 0.25 ⁇ M. Place each qPCR reaction system containing the target gene HSD on the ABI StepOnePlus Real-Time PCR instrument and use a three-step method for amplification.
  • the amplification program is pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 30 seconds, and denaturation at 55°C for 30 seconds.
  • Anneal for 30 seconds extend at 72°C for 30 seconds, and repeat the above-mentioned denaturation, annealing, and extension processes for a total of 40 times to obtain the product W containing the amplified target gene HSD; for the qPCR system containing the internal reference gene GAPDH, use the same method as above Perform qPCR amplification, the only difference is that the annealing temperature in the amplification program is 60°C, and the product W' containing the amplified internal reference gene GAPDH is obtained.
  • the products W and W' were then incubated at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.
  • the dissolution curves of the target gene HSD and the internal reference gene GAPDH in the products W and W' were measured using a real-time fluorescence quantitative PCR instrument to obtain the target. Ct values of gene HSD and internal reference gene GAPDH.
  • the comparative Ct ( ⁇ Ct) method was used to calculate the relative quantification of HSD of the target gene in each test group.
  • ⁇ Ct average control group
  • ⁇ Ct control group
  • HSD mRNA expression level in the test group was normalized, and the HSD mRNA expression level in the control group was defined as 100%.
  • Relative expression level of HSD mRNA in the test group 2 - ⁇ Ct (test group) ⁇ 100%
  • Inhibition rate of HSD mRNA in the test group (1-relative expression level of HSD mRNA in the test group) ⁇ 100%
  • Table 5 shows the inhibition rate of HSD mRNA in monkey primary hepatocytes after transfection of siRNA Conjugate 6 and Conjugate 7 of the present disclosure.
  • siRNA Conjugate 6 and Conjugate 7 provided by the present disclosure have the same sequence as the reference but do not contain stabilizing modified nucleotides at a concentration of 50 nM. showed significantly higher HSD mRNA inhibitory activity compared to the conjugates.
  • Monkey primary hepatocytes were prepared using the same method as in Experimental Example 2.
  • Each 3A solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 2 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 3Aa and 3Ab respectively.
  • Each 3B solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 3 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 3Ba and 3Bb respectively.
  • Each 3C solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 5 working solution and 97 ⁇ l of cell maintenance medium in order to obtain 3Ca and 3Cb siRNA working solutions respectively.
  • Each 3D solution contains 3 ⁇ l of the above reference conjugate NC working solution and 97 ⁇ l of cell maintenance medium in order to obtain 3Da and 3Db siRNA working solutions respectively.
  • Each 3E1 solution contains 2 ⁇ l Lipofectamine TM 2000 and 98 ⁇ l cell maintenance medium.
  • siRNA conjugate 2 In the two culture wells, add the transfection complex 3Xa1 or 3Xa2 of siRNA conjugate 2 respectively, and mix evenly. The addition amount is 200 ⁇ l/well, and the final concentration of each siRNA conjugate (calculated as siRNA) is approximately 50 nM. or 5nM transfection mixture, recorded as test group 1.
  • siRNA conjugate 3 In the two culture wells, add the transfection complex 3Xb1 or 3Xb2 of siRNA conjugate 3 respectively, and mix evenly. The addition amount is 200 ⁇ l/well, and the final concentration of each siRNA conjugate (calculated as siRNA) is approximately The transfection mixture of 50nM or 5nM was recorded as test group 2.
  • siRNA conjugate 5 In the two culture wells, add the transfection complex 3Xc1 or 3Xc2 of siRNA conjugate 5 respectively, and mix evenly. The addition amount is 200 ⁇ l/well, and the final concentration of each siRNA conjugate (calculated as siRNA) is approximately The transfection mixture of 50nM or 5nM was recorded as test group 3.
  • the cells containing siRNA conjugate 2, conjugate 3, conjugate 5 and reference conjugate NC were The 12-well plate of the transfection mixture was placed in an incubator containing 5% CO 2 /95% air and continued to be cultured at 37°C for 24 hours.
  • Figure 1 is a bar graph showing the relative expression levels of HSD mRNA in monkey primary hepatocytes in vitro after transfection with 50 nM and 5 nM concentrations of siRNA of the present disclosure.
  • blank represents the blank control group
  • NC represents the negative control group.
  • siRNA Conjugate 2, Conjugate 3 and Conjugate 5 provided by the present disclosure showed high HSD mRNA inhibition at siRNA concentrations of 50nM and 5nM. active.
  • target sequence 4 contains a sequence that is partially complementary to the siRNA antisense strand in the conjugate to be tested, so the inhibitory effect of the conjugate to be tested on target sequence 4 can reflect the degree of off-target effects. That is, the higher the inhibitory effect, the more likely the test conjugate will be off-target.
  • siRNA conjugate 3 of the present disclosure has While greatly improving HSD mRNA inhibitory activity, it also exhibits lower off-target effects than the reference conjugate, and has excellent drug-making capabilities.
  • Mouse liver primary cells were extracted from fresh liver tissue of C57BL/6j mice, and the density of primary mouse liver cells was adjusted to 1 ⁇ 10 in Opti-MEM (1X) medium (GIBCO Company, Cat. No. 31985-070) 5 cells/mL to obtain mouse liver primary cell suspension. Then, a volume of 1 mL/well of mouse liver primary cell suspension was added to different culture wells of the 12-well plate.
  • Opti-MEM (1X) medium GEBCO Company, Cat. No. 31985-070
  • Each 5A solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 6 working solution and 97 ⁇ l of cell maintenance medium in order to obtain 5A1 siRNA working solution.
  • Each 5B solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 7 working solution and 97 ⁇ l of cell maintenance medium in order to obtain 5B siRNA working solution.
  • Each 5C solution contains 3 ⁇ l of the above reference conjugate NC working solution and 97 ⁇ l of cell maintenance medium in order to obtain 5C siRNA working solutions.
  • each 5D solution contains 2 ⁇ l Lipofectamine TM 2000 and 98 ⁇ l cell maintenance medium.
  • transfection complex 5Xa of siRNA Conjugate 6 In two culture wells, add transfection complex 5Xa of siRNA Conjugate 6 respectively, mix evenly, and add 200 ⁇ l/well to obtain a final concentration of siRNA Conjugate 6 (based on siRNA) of approximately 50 nM for transfection.
  • the compound is designated as test group 1.
  • siRNA Conjugate 7 In the two culture wells, add the transfection complex 5Xb of siRNA Conjugate 7 respectively, mix evenly, and the addition amount is 200 ⁇ l/well to obtain the final concentration of siRNA Conjugate 7 (based on siRNA (calculated) were approximately 50 nM of the transfection complex, recorded as test group 2.
  • siRNA reference conjugate NC transfection complex 5Xc In the two culture wells, add siRNA reference conjugate NC transfection complex 5Xc respectively, mix evenly, the addition amount is 200 ⁇ l/well, and the final concentration of control reference conjugate NC (based on siRNA) is approximately 50nM transfection complex was recorded as the negative control group.
  • cell maintenance medium was added at an amount of 200 ⁇ l/well, which was recorded as the blank control group.
  • siRNA-containing transfection mixture and blank control group were placed in an incubator containing 5% CO2 and continued to be cultured at 37°C for 24 h.
  • Figure 2 is a bar graph showing the relative expression levels of HSD mRNA in primary hepatocytes of C57BL/6j mice after conjugate 6, conjugate 7 or reference conjugate NC.
  • blank represents the blank control group
  • lipo represents the Lipo control group
  • NC represents the negative control group.
  • the siRNA conjugate of the present disclosure shows excellent HSD mRNA inhibition activity in C57BL/6j mouse primary liver cells. At a siRNA concentration of 50nM, the HSD mRNA inhibition rate is at least 83.11%.
  • Conjugate 8 and reference conjugate 7 were dissolved in PBS to a solution of 3 mg/ml (based on siRNA conjugate).
  • C57BL/6 mice female, 16-18 g weight, 6-8 weeks old, purchased from (Usperford Co., Ltd.) were randomly divided into groups, with 6 mice in each group and numbered respectively.
  • the administration volume is 5 mL/kg, which serves as the test group; in addition, PBS was administered to each mouse in a group at a volume of 5 mL/kg, serving as a blank control group.
  • mice are expressed as mean ⁇ standard deviation (Mean ⁇ SD), and data analysis uses Graphpad prism statistical analysis software. If the data conforms to the normal distribution and the variances are equal, the one-way ANOVA method is used for testing; if the data does not conform to the normal distribution or the variances are uneven, the Kruskal-Wallis H method of the non-parametric test is used for analysis, P ⁇ 0.05 is considered a statistically significant difference.
  • Figure 3 shows the inhibitory effect of conjugate on HSD17B13 mRNA in mouse hepatocytes on day 8 after administration of 3 mg/kg of conjugate 8, reference conjugate 7 or PBS.
  • Figure 3 shows that compared with the blank control group, after administration of reference conjugate 7 that does not contain stabilizing modified nucleotides, the inhibition rate of HSD17B13 mRNA in mouse liver cells was 57.2%; while administration of the siRNA conjugate of the present disclosure After conjugation, the inhibition rate of HSD17B13 mRNA in mouse hepatocytes was 69.34%, showing higher inhibitory activity than the blank control group and reference conjugate 7.
  • Each 7A solution contains 3 ⁇ l of the above-mentioned siRNA Conjugate 1 working solution and 97 ⁇ l of cell maintenance medium.
  • Each 7B solution contains 2 ⁇ l Lipofectamine TM 2000 and 98 ⁇ l cell maintenance medium.
  • transfection complex 7X a of siRNA Conjugate 1 In the two culture wells, add the transfection complex 7X a of siRNA Conjugate 1 respectively, mix evenly, and add 200 ⁇ l/well to obtain a final concentration of siRNA Conjugate 1 (based on siRNA) of about 50 nM.
  • the transfection mixture of transfection complexes was designated as test group 1.
  • the 12-well plate containing the transfection mixture containing siRNA conjugate 1 was placed in an incubator containing 5% CO 2 /95% air and continued to be cultured at 37°C for 24 hours.
  • siRNA conjugate 1 containing stabilized modified nucleotides provided by the present disclosure has an inhibition rate of HSD mRNA in monkey primary liver cells at a concentration of 50 nM reaching 92.47%, showing excellent inhibitory activity.
  • Conjugate 3 was dissolved in PBS to a solution of 0.6 mg/ml (based on siRNA conjugate).
  • SD rats male, 200-240g weight, 6-8 weeks old, purchased from Spefford Company
  • the administration volume is 5 mL/kg, which serves as the test group;
  • PBS was administered to each mouse in a group of mice at a volume of 5 mL/kg, serving as a blank control group.
  • the rat internal reference gene is ACTIN, and the primer information used is as follows in Table 8. shown.
  • siRNA conjugate 3 conjugate 5, conjugate 7, conjugate 8, conjugate 9, conjugate 10 and reference conjugate NC were formulated with DEPC water to 20 ⁇ M and 2 ⁇ M respectively (in siRNA siRNA conjugate working solution.
  • Each 9A solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 3 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 9A 1 and 9A 2 respectively.
  • Each 9B solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 5 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 9B 1 and 9B 2 respectively.
  • Each 9C solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 7 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 9C 1 and 9C 2 respectively.
  • Each 9D solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 8 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 9D 1 and 9D 2 respectively.
  • Each 9E solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 9 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 9E 1 and 9E 2 respectively.
  • Each 9F solution contains 3 ⁇ l of the above-mentioned siRNA conjugate 10 working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 9F 1 and 9F 2 respectively.
  • Prepare 9G solution each 9G solution contains 2 ⁇ l Lipofectamine TM 2000 and 98 ⁇ l cell maintenance medium.
  • Prepare a 9H solution Each 9H solution contains 3 ⁇ l of the above reference conjugate NC working solution and 97 ⁇ l of cell maintenance medium in order to obtain siRNA working solutions of 9H 1 and 9H 2 respectively.
  • transfection complex 9X a1 or 9X a2 of siRNA Conjugate 3 respectively, mix evenly, and add 200 ⁇ l/well to obtain a final concentration of siRNA Conjugate 3 (based on siRNA) of approximately
  • the transfection mixture is 50nM or 5nM, recorded as test group 1.
  • transfection complex 9X b1 or 9X b2 of siRNA Conjugate 5 respectively, mix evenly, and add 200 ⁇ l/well to obtain a final concentration of siRNA Conjugate 5 (based on siRNA) of approximately
  • the transfection mixture is 50nM or 5nM, recorded as test group 2.
  • transfection complex 9X c1 or 9X c2 of siRNA conjugate 7 respectively, mix evenly, and add an amount of 200 ⁇ l/well to obtain the final concentration of reference siRNA conjugate 7 (calculated as siRNA). ) is about 50nM or 5nM transfection mixture, recorded as test group 3.
  • transfection complex 9X d1 or 9X d2 of siRNA conjugate 8 respectively, mix evenly, and add 200 ⁇ l/well to obtain the final concentration of reference siRNA conjugate 8 (calculated as siRNA). ) is about 50nM or 5nM transfection mixture, recorded as test group 4.
  • transfection complex 9X e1 or 9X e2 of siRNA conjugate 9 respectively, mix evenly, and add 200 ⁇ l/well to obtain the final concentration of reference siRNA conjugate 9 (calculated as siRNA). ) is about 50nM or 5nM transfection mixture, recorded as test group 5.
  • transfection complex 9X f1 or 9X f2 of siRNA conjugate 10 are added evenly, and add an amount of 200 ⁇ l/well to obtain the final concentration of reference siRNA conjugate 10 (calculated as siRNA). ) is approximately 50 nM or 5 nM of the transfection mixture, recorded as test group 6.
  • siRNA concentration of reference siRNA conjugate 10
  • 2X g of blank transfection complex respectively at an amount of 200 ⁇ l/well to obtain a transfection mixture without siRNA, which is recorded as the blank control group.
  • a 12-well plate containing the transfection mixture containing siRNA conjugate 3, conjugate 5, conjugate 7, conjugate 8, conjugate 9, conjugate 10 and reference conjugate NC was placed. Continue culturing for 24 hours at 37°C in an incubator containing 5% CO 2 /95% air.
  • the siRNA conjugates provided by the present disclosure include conjugates containing 1 base mismatch and conjugates not containing 1 base mismatch. Exhibits excellent HSD mRNA inhibitory activity.
  • siRNA conjugate 3 conjugate 5, conjugate 7, conjugate 8, conjugate 9 and conjugate 10 were formulated with DEPC water to 0.12 ⁇ M and 0.012 ⁇ M (based on siRNA) siRNA conjugates, respectively.
  • Compound working fluid Compound working fluid.
  • each 10A solution contains 2 ⁇ l Lipofectamine TM 2000 and 98 ⁇ l cell maintenance medium.
  • each siRNA conjugate (based on siRNA) of approximately 10 nM or 10 nM respectively.
  • the 1 nM co-transfection mixture was designated as test group 2.
  • siRNA conjugate 7 transfection complex 10X 3 or 10X 03 mix evenly, and add 200 ⁇ l/well.
  • a co-transfection mixture with a final concentration of each siRNA conjugate (based on siRNA) of approximately 10 nM or 1 nM was obtained, which was recorded as test group 3.
  • siRNA conjugate 8 transfection complex 10X 4 or 10X 04 mix evenly, and add 200 ⁇ l/well.
  • a co-transfection mixture with a final concentration of each siRNA conjugate (based on siRNA) of approximately 10 nM or 1 nM was obtained, which was recorded as test group 4.
  • siRNA conjugate 9 transfection complex 10X 5 or 10X 05 mix evenly, and add 200 ⁇ l/well.
  • a co-transfection mixture with a final concentration of each siRNA conjugate (based on siRNA) of approximately 10 nM or 1 nM was obtained, which was recorded as test group 5.
  • siRNA conjugate 10 transfection complex 10X 6 or 10X 06 mix evenly, and add 200 ⁇ l/well.
  • a co-transfection mixture with a final concentration of each siRNA conjugate (based on siRNA) of approximately 10 nM or 1 nM was obtained, which was recorded as test group 6.
  • a 12-well plate containing the transfection mixture containing siRNA conjugate 3, conjugate 5, conjugate 7, conjugate 8, conjugate 9 and conjugate 10 was placed in a 5% CO 2 / The culture was continued in an incubator with 95% air at 37°C for 24 h.
  • the detection plasmid and the conjugate to be tested are co-transfected into HEK293A cells, and the level of SEAP enzyme activity is used to reflect the target sequence inhibitory activity of siRNA.
  • the above-mentioned SEAP-HSD plasmid contains the HSD cDNA sequence (Genbank registration number NM_001136230.3) inserted into the 3'UTR of the SEAP (secreted alkaline phosphatase, purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.) reporter gene.
  • SEAP Reporter GENE ASSAY KIT Luminescence abcam-ab 133077Protocol measured the above cells.
  • Figure 5 shows a bar graph of SEAP concentration in HEK293A cells at 10 nM and 1 nM concentrations.
  • conjugate 9 showed a particularly high inhibition rate of HSD mRNA at 72 h, with an inhibition rate of 91.6% at a concentration of 10 nM and 89.9% at a concentration of 1 nM.
  • the inhibitory activity on HSD17B13 gene expression mRNA can reach up to 92.4% at 72 hours.
  • the inhibition rate of each group at 72 hours is generally higher than the inhibition rate at 24 hours, showing long-lasting inhibitory activity on HSD17B13 mRNA.
  • SD rats Male, 200-240g weight, 6-8 weeks old, purchased from Spefford Company were randomly divided into 3 groups, 5 rats in each group.
  • the reference conjugate 3 and conjugate 9 prepared above were prepared into 0.6 mg/mL solutions (based on siRNA) in PBS.
  • the above siRNA conjugate solution was administered to each group of mice by subcutaneous injection on the back of the neck. Weigh and record the body weight before administration. Administration was based on body weight. The volume of a single administration was 5 ⁇ L/g mouse body weight. Calculate It can be seen that the single dose is 3mg/kg.
  • PBS was administered to each mouse in one group, with a single administration volume of 5 ⁇ L/g, serving as a blank control group.
  • Figure 6 shows the inhibition rate of HSD17B13 mRNA in mouse hepatocytes by the conjugate on days 8 and 15 after administration of 3 mg/kg of siRNA conjugate of the present disclosure.
  • “Comparative Conjugate 3” represents the results of the reference conjugate 3
  • “PBS” represents the results of the blank control group.
  • the results of Figure 6 show that after administration of the siRNA conjugates containing stabilized modified nucleotides of the present disclosure, the inhibition rate of HSD17B13 mRNA in mouse liver cells was significantly higher than that of the reference conjugate, showing higher inhibitory activity. .
  • the inhibition rate of HSD17B13 mRNA was 81.2%.
  • SD rats purchased from Spefford (Beijing) Biotechnology Co., Ltd. were divided into 5 groups, with 10 rats in each group, half male and half female.
  • Rats in each group were subcutaneously administered a single dose of 30 mg/kg or 100 mg/kg (calculated as siRNA) of Conjugate 3, Conjugate 9 and PBS of the present disclosure as the control group. They were weighed and recorded before administration. The drugs were administered according to body weight, and the day of administration was recorded as D1. The drugs were administered on D15 and D29 respectively for a total of 3 times. Histopathological examination was performed on D30.
  • the rats showed no death or behavioral abnormalities, and there was no significant difference in weight gain between the animals in each dose group and the control group.
  • liver pathological examination The rats were dissected, and the liver tissue was dissected. After sampling, dehydration, embedding, making and staining, pathological sections were made and examined under an optical microscope (microscope model: NIKON Eclipse ci, imaging system: NIKON digital sight DS-FI2, MADE IN JAPAN) observation.
  • NIKON Eclipse ci imaging system: NIKON digital sight DS-FI2, MADE IN JAPAN
  • the siRNA conjugate of the present disclosure has a more effective effect on inhibiting HSD17B13 mRNA, and therefore shows significant potential in the preparation of drugs for the treatment and/or prevention of HSD diseases or symptoms. Higher therapeutic effect and excellent development prospects.
  • any combination of various embodiments of the present disclosure can also be carried out, and as long as they do not violate the idea of the present disclosure, they should also be regarded as the contents disclosed in the present disclosure.

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Abstract

提供了一种能够抑制17β-羟基类固醇脱氢酶13型(HSD17B13)基因表达的siRNA，该siRNA包含正义链和反义链，该正义链和反义链分别包含核苷酸序列I和核苷酸序列II，该核苷酸序列I和核苷酸序列II分别由19个修饰或未修饰的核苷酸组成，并至少部分地反向互补形成双链区，该核苷酸序列II至少部分地与HSD17B13基因表达的mRNA中的一段核苷酸序列反向互补；按照5'末端到3'末端的方向，该核苷酸序列II的第3-6个核苷酸中的至少1个为稳定化修饰核苷酸。该siRNA或包含该siRNA的药物组合物或siRNA缀合物可以有效治疗和/或预防与HSD17B13基因表达相关的疾病或病症。

Description

一种核酸、含有该核酸的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17β-羟基类固醇脱氢酶13型(HSD17B13或17beta-HSD13)基因表达的核酸和含有该核酸的组合物与缀合物。本公开还涉及这些核酸、组合物与缀合物的制备方法和用途。
背景技术
17β-羟基类固醇脱氢酶13型(通常称为HSD17B13或17beta-HSD13)是17β-羟基类固醇脱氢酶(17β-HSD)家族的成员。17β-羟基类固醇脱氢酶13型是位于肝细胞脂滴表面的一种蛋白，其功能可能与肝脏脂质代谢以及转氨酶的生成有关，已知在肝脏的肝细胞中发现了很高的表达水平，而在卵巢、骨髓、肾脏、脑、肺、骨骼肌、膀胱和睾丸中可以检测到较低的水平。
研究证明，HSD17B13的水平在脂肪肝或糖尿病小鼠肝脏高表达，在C57BL/6鼠体内的肝脏过度表达显著地增加肝脏中的脂肪生成及甘油三酸酯(TG)含量，导致脂肪肝表型。目前，HSD17B13已被鉴定为非酒精性脂肪肝病(NAFLD)患者中的脂质小滴(LD)相关蛋白。过表达的HSD17B13会导致LD的数目和大小的增加，而脂质小滴(LD)蓄积与多种代谢疾病及慢性纤维炎性肝病如：肝纤维化、非酒精性脂肪性肝炎(NASH)及NAFLD相关。表达HSD17B13的基因的敲除减弱了培养的肝细胞中油酸诱导的LD形成。已有报道表明，在脂肪肝患者中已观察到肝脏中HSD17B13上调，这支持了HSD17B13在NAFLD的发病机制中的作用。
目前，在慢性纤维性炎性肝病治疗领域中，对于罹患慢性纤维炎性肝病的个体的疗法仍存在未被满足的需求。
发明内容
为了开发一种能够抑制HSD17B13基因的siRNA，发明人发现，在序列中引入具有稳定化修饰核苷酸的siRNA出人意料地显示出比对应位置不具有稳定化修饰核苷酸的siRNA显著更高的HSD17B13基因抑制活 性，因此，发明人作出如下发明。
在一方面，本公开提供了一种siRNA，所述siRNA包含正义链和反义链，所述正义链包含核苷酸序列I，所述反义链包含核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II均由19个核苷酸组成，所述核苷酸序列I和所述核苷酸序列II中的每一个核苷酸均为修饰或未修饰的核苷酸，所述核苷酸序列I和所述核苷酸序列II至少部分地与第一段核苷酸序列反向互补，所述第一段核苷酸序列为HSD17B13基因表达的mRNA中的一段长度为19个核苷酸的核苷酸序列，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-6个核苷酸中的至少1个为稳定化修饰核苷酸，所述稳定化修饰核苷酸指核苷酸的核糖2'位羟基被稳定化修饰基团取代的核苷酸，与相应位置的核苷酸为未修饰的核苷酸的siRNA相比，包含所述稳定化修饰核苷酸的siRNA的热稳定性增加，并且所述稳定化修饰基团的空间位阻大于2'-O-甲基。
在另一方面，本公开还提供了一种药物组合物，该药物组合物含有本公开提供的siRNA以及药学上可接受的载体。
在又一方面，本公开还提供了一种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，所述缀合基团包含接头和药学上可接受的靶向基团，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，每个所述靶向基团选自能够和细胞表面受体结合的配体。
在又一方面，本公开还提供了本公开的siRNA和/或本公开的药物组合物和/或本公开的siRNA缀合物在制备用于治疗和/或预防与HSD17B13基因表达的mRNA水平相关的疾病或者症状的药物中的用途。
在又一方面，本公开还提供了一种治疗和/或预防与HSD17B13基因表达的mRNA水平相关的疾病或症状的方法，所述方法包括向有需要的受试者给予本公开的siRNA，和/或本公开的药物组合物，和/或本公开的siRNA缀合物。
在又一方面，本公开还提供了一种抑制细胞中HSD17B13基因表达水平的方法，所述方法包括将有效剂量的本公开的siRNA，和/或本公开 的药物组合物和/或本公开的siRNA缀合物与所述细胞接触。
在又一方面，本公开还提供了一种试剂盒，所述试剂盒包含本公开的siRNA，和/或本公开的药物组合物，和/或本公开的siRNA缀合物。
以引用的方式并入
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本文，其程度与每一单独的出版物、专利或专利申请均专门并且单独地以引用的方式并入本文的程度相同。
有益效果
本公开的siRNA、药物组合物和/或siRNA缀合物具有较高的HSD17B13基因表达的抑制活性和/或低的毒性，能够有效治疗或预防与HSD17B13基因表达的mRNA水平相关的疾病或症状。
本公开的siRNA、药物组合物和/或siRNA缀合物显示出优异的HSD17B13基因表达的mRNA的调节活性。例如，本公开提供的siRNA缀合物在psi-CHECK系统中有很高的目标序列抑制活性，与其序列相同、但不包含稳定化修饰核苷酸的参比缀合物相比，本公开的缀合物对目标序列抑制活性大大提高，IC50值仅为参比缀合物1的约1/4至1/3。
又例如，在猴原代肝细胞中，本公开提供的siRNA缀合物在50nM或者5nM浓度下，HSD17B13基因表达的mRNA的抑制活性至少为91.30％，甚至可高达94.71％，与不包含稳定化修饰核苷酸的参比缀合物相比，本公开的缀合物均表现出更加优异的HSD17B13 mRNA抑制活性。又例如，在猴原代肝细胞中，本公开提供的siRNA缀合物在10nM浓度下，对HSD17B13基因表达的mRNA的抑制活性最高可达到92.4％。又例如，在猴原代肝细胞中，本公开提供的siRNA缀合物在50nM或者5nM浓度下，均表现出优异的HSD17B13 mRNA抑制活性，50nM浓度明显高于5nM浓度的抑制活性，最高可达98％。
又例如，在小鼠体内，本公开提供的siRNA缀合物HSD17B13 mRNA的抑制率为69.34％，与给予不包含稳定化修饰核苷酸的参比缀合物相比，本公开的缀合物均表现出更加优异的HSD17B13 mRNA抑制活性。又例如，在大鼠体内，给予包含稳定化修饰核苷酸的本公开的siRNA缀合物 后，在第8天时，HSD17B13 mRNA的抑制率为81.2％，与不包含稳定化修饰核苷酸的参比缀合物相比，表现出更加优异的HSD17B13 mRNA抑制活性；在第15天时更是显著高于对比缀合物，表明本公开的siRNA缀合物还具有优异的体内HSD17B13mRNA长效抑制活性。
实验结果证明，在大鼠体内，给予最高达100mg/kg剂量的本公开的siRNA缀合物后，各剂量组的组织结构和脏器重量均无显著性改变，显示出本公开的缀合物毒性小，体内耐受性良好，具有30倍以上的安全窗口，适合作为体内抑制HSD17B13mRNA的药物使用。
进一步地，本公开提供的siRNA缀合物在psi-CHECK系统中对脱靶序列的抑制率在测试浓度范围内均不高于50％，显示出与不包含稳定化修饰核苷酸的参比缀合物相比更低的脱靶效应。
综上所述，本公开提供的siRNA、药物组合物以及siRNA缀合物能够有效抑制HSD17B13基因的表达，在保证安全性的剂量下，有效治疗和/或预防由HSD17B13基因表达的mRNA水平相关的疾病症状，具有良好的应用前景。
附图说明
附图是用来提供对本公开的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本公开，但并不构成对本公开的限制。在附图中：
图1是显示了转染50nM和5nM浓度的本公开的siRNA后，体外猴原代肝细胞中的HSD mRNA相对表达水平的柱状图。
图2是显示了转染50nM浓度的本公开的siRNA后，体外C57BL/6j小鼠原代肝细胞中的HSD mRNA相对表达水平的柱状图。
图3是显示了在给予本公开的3 mg/kg的siRNA后，小鼠体内的HSD mRNA相对表达水平的图。
图4是显示了转染50nM和5nM浓度的本公开的siRNA后，体外猴原代肝细胞中的HSD mRNA相对表达水平的柱状图。
图5是显示了转染10nM和1nM浓度的本公开的siRNA后，24小 时和72小时体外猴原代肝细胞中SEAP浓度的折线图。
图6是显示了在给予本公开的3 mg/kg的siRNA和参比缀合物后，小鼠体内的HSD mRNA相对表达水平的图。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
在本公开中，如无其他说明，HSD17B13 mRNA或“HSD17B13基因表达的mRNA”是指具有如Genbank注册号NM_001136230.3所示序列的mRNA，在上下文中也简称为HSD mRNA，HSD17B13基因是指转录上述HSD17B13 mRNA的基因。
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，在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。
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，特别是在描述本公开的siRNA、药物组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)是指，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。
在上文或下文中，“经取代的”或“被取代的”基团，如经取代的烷基、经取代的烷氧基、经取代的氨基、经取代的脂族基团、经取代的杂脂族基团、经取代的酰基、经取代的芳基或经取代的杂芳基。其中，如无其他说明，“经取代的”或“被取代的”基团是指该基团中的氢原子被一个或多个取代基所替代而形成的基团。例如，“经取代的烷氧基”是指烷氧基中的一个或多个氢原子被取代基所替代而形成的基团。本领域技术人员能够理解，可用于本公开应用的化合物中可以包含各种取代基， 只要是该取代基的引入不会影响本公开的功能，能够实现本公开的目的，就可用于本公开。在一些实施方式中，所述取代基选自于由以下基团所组成的组：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、-CN、-NO2、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。在一些实施方式中，所述取代基是C1-C3烷基、C6-C8芳基、-OC1-C3烷基、-OC1-C3烷基苯基、卤素、-OH、-NH2、-CN或-NO2中的一种。本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或本身不稳定的任何取代或取代模式。
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链，所述数量通常为1至20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C1-C6烷基包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1- 烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1,3-二烯-1-基、丁-1,3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。
如本文所使用的，“炔基”是指具有至少一个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两分子氢而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实施方式中，炔基具有2到20个碳原子，而在其他实施方式中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个连接点的残基。
如本文所使用的，“烷氧基”是指通过氧桥连接的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8个、1至6个，或1至4个通过氧桥连接的碳原子。
如本文所使用的，“芳基”是指通过从环碳原子中除去氢原子而衍生自芳香族单环或多环烃环系统形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的至少一个环是完全不饱和的，即，包含根据Hückel理论的环状、离域的(4n+2)π-电子体系。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的子集，指与芳基相同、但具有两个连接点的残基。
“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。在一些实施方式中，杂芳基中的杂原子是氧化的杂原子。在一些实施方式中，杂芳基中包含一个或多个氮原子。在一些实施方式中，杂芳基中的氮原子中的一个或多个是季铵化 的氮原子。杂芳基通过任何环原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1,3-苯并二噁唑基、苯并呋喃基、苯并噁唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1,4]二噁庚英基(benzo[b][1,4]dioxepinyl)、苯并[b][1,4]噁嗪基(benzo[b][1,4]oxazinyl)、1,4-苯并二噁烷基(1,4-benzodioxanyl)、苯并萘并呋喃基、苯并噁唑基、苯并间二氧杂环戊烯基(benzodioxolyl)、苯并二噁英基(benzodioxinyl)、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩并[3,2-d]嘧啶基、苯并三唑基、苯并[4,6]咪唑并[1,2-a]吡啶基、咔唑基、噌啉基(cinnolinyl)、环戊烷并[d]嘧啶基、6,7-二氢-5H-环戊烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6-二氢苯并[h]喹唑啉基(5,6-dihydrobenzo[h]quinazolinyl)、5,6-二氢苯并[h]噌啉基(5,6dihydrobenzo[h]cinnolinyl)、6,7-二氢-5H-苯并[6,7]环庚烷并[1,2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃并[3,2-c]吡啶基、5,6,7,8,9,10-六氢环辛烷并[d]嘧啶基、5,6,7,8,9,10-六氢环辛烷并[d]哒嗪基、5,6,7,8,9,10-六氢环辛烷并[d]吡啶基、异噻唑基、咪唑基、吲唑基(indazolyl)、吲哚基、异吲哚基、二氢吲哚基、异二氢吲哚基、异喹啉基、吲哚嗪基(indolizinyl)、异噁唑基、5,8-甲醇-5,6,7,8-四氢喹唑啉基(5,8-methano-5,6,7,8-tetrahydroquinazolinyl)、萘啶基(naphthyridinyl)、1,6-萘啶酮基(1,6-naphthyridinonyl)、噁二唑基、2-氧杂吖庚因基(2-oxoazepinyl)、噁唑基、氧杂环丙烷基(oxiranyl)、5,6,6a,7,8,9,10,10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基(phthalazinyl)、蝶啶基(pteridinyl)、嘌呤基、吡咯基、吡唑基、吡唑并[3,4-d]嘧啶基、吡啶基、吡啶并[3,2-d]嘧啶基、吡啶并[3,4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基(quinoxalinyl)、喹啉基、四氢喹啉基、5,6,7,8-四氢喹唑啉基、5,6,7,8-四氢苯并[4,5]噻吩并[2,3-d]嘧啶基、6,7,8,9-四氢-5H-环庚烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6,7,8-四氢吡啶并[4,5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩并[2,3-d]嘧啶基、噻吩并[3,2-d]嘧啶基、噻吩并[2,3-c]吡啶基(thieno[2,3-c]pridinyl)和噻吩基(thiophenyl/thienyl)。
如本文所使用的，“卤素取代基”或“卤代”指氟代、氯代、溴代和碘代，术语“卤素”包括氟、氯、溴和碘。
如本文所使用的，“卤代烷基”是指指定数量的氢原子被一个或多个、直至最大允许数量的卤素原子取代的如上述所定义的烷基。卤代烷基的实例包括但不限于三氟甲基、二氟甲基、2-氟乙基和五氟乙基。
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能团对特定的反应条件不敏感，并且可以在分子中的该官能团上添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2,2d ed，John Wiley & Sons，New York，1991中，以引用的方式将上述文献各自整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基氧杂蒽-9-基(Pixyl)和9-(对甲氧基苯基)氧杂蒽-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4'-二甲氧基三苯甲基)和TMTr(4,4',4”-三甲氧基三苯甲基)。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、兔、绵羊、大鼠和任何种类的家禽。
如本文所使用的，“治疗”指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。
如本文所使用的“预防”指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将双链siRNA、药物组合物或siRNA缀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。
本公开所述siRNA或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子，铵离子NH4 +，有机铵阳离子中的一种，处于提高溶解性考虑，在一些实施方式中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属阳离子可以是K+和/或Na+，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N,N-二异丙基乙胺形成的铵离子。因此，本公开所述siRNA或siRNA缀合物可以至少部分以盐的形式存在。在一些实施方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本公开所述siRNA或siRNA缀合物以钠盐或部分钠盐的形式存在。因此，在提及本公开所述的siRNA或siRNA缀合物，包括但不限于本公开所述的任何结构式表示的siRNA缀合物时，均旨在涵盖该siRNA或siRNA缀合物的钠盐或部分钠盐形式。
本公开的siRNA
在一方面，本公开提供了一种具有较高的HSD17B13基因抑制活性的siRNA。
本公开的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基，在此不再赘述。
本公开的siRNA包含正义链和反义链，所述正义链包含核苷酸序列I，所述反义链包含核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II均由19个核苷酸组成，所述核苷酸序列I和所述核苷酸序列II中的每一个核苷酸均为修饰或未修饰的核苷酸，所述核苷酸序列I和所述核苷酸序列II至少部分地与第一段核苷酸序列反向互补，所述第一段核苷酸序列为HSD17B13基因表达的mRNA中的一段长度为19个核苷酸的核苷酸序列，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-6个核苷酸中的至少1个为稳定化修饰核苷酸，所述稳定化修饰核苷酸指核苷酸的核糖2'位羟基被稳定化修饰基团取代的核苷酸，与相应位置的核 苷酸为未修饰的核苷酸的siRNA相比，包含所述稳定化修饰的核苷酸siRNA的热稳定性增加，并且所述稳定化修饰基团的空间位阻大于2'-O-甲基。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第3个或第5个核苷酸为所述稳定化修饰核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第3-9个核苷酸中不超过2个核苷酸为所述稳定化修饰核苷酸。通过对特定位置处稳定化修饰核苷酸的个数进行限定，本公开的siRNA可获得最佳的药学活性与低的脱靶效应的平衡，同时还具有优异的稳定性。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第3个和/或第5个核苷酸为所述稳定化修饰核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第3个核苷酸为所述稳定化修饰核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第5个核苷酸为所述稳定化修饰核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中第3-9个核苷酸之外的核苷酸均不是稳定化修饰核苷酸。若核苷酸序列II中在第3-6个核苷酸中的至少1个为稳定化修饰的核苷酸的同时，在第3-9个核苷酸之外包含稳定化修饰核苷酸，可能会显著影响该siRNA的目标序列表达水平的调节能力。
在一些实施方式中，本公开的上下文中“siRNA的热稳定性增加”是指所述siRNA的热解离温度(Tm)升高。在一些实施方式中，“双链siRNA的热稳定性增加”是指siRNA的Tm升高至少0.05℃，在一些实施方式中指升高0.1-6℃，在一些实施方式中指Tm升高0.5-4℃。通过在特定位置包含稳定化修饰核苷酸，本公开的siRNA出人意料地表现出更高的HSD17B13 mRNA抑制活性。在一些实施方式中，本公开的siRNA还显示出与不包含稳定化修饰核苷酸的siRNA相比更低的脱靶效应。
在一些实施方式中，每个所述稳定化修饰基团独立地具有-X-R所示的结构，其中，X为O、NR'、S或SiR'2；R是C2-C6烷基、取代的C2-C6烷基、C6-C8芳基、取代的C6-C8芳基中的一种，每个R'独立地是H、C1-C6烷基、取代的C1-C6烷基、C6-C8芳基、取代的C6-C8芳基中的一种，所 述取代的C2-C6烷基、取代的C6-C8芳基或取代的C1-C6烷基指C2-C6烷基、C6-C8芳基或C1-C6烷基中的一个或多个氢原子被取代基团取代而形成的基团，所述取代基团选自以下取代基中的一种或多种：C1-C3烷基、C6-C8芳基、C1-C3烷氧基、卤素、氧亚基和硫亚基。注意的是，本公开并非旨在涵盖全部符合上述结构的修饰基团，而仅涉及哪些能够实现siRNA热稳定性增加的稳定化修饰基团，在一些实施方式中，每个所述稳定化修饰基团独立地选自2'-O-甲氧基乙基、2'-O-烯丙基、2'-烯丙基、2'-O-2-N-甲基氨基-2-氧亚基乙基、2'-O-2-N,N-二甲基氨基乙基、2'-O-3-氨基丙基和2'-O-2,4-二硝基苯基中的一种。在一些实施方式中，每个所述稳定化修饰基团均为2'-O-甲氧基乙基。
在一些实施方式中，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，所述反义链的长度为19-26个核苷酸，这样，本公开提供的siRNA正义链和反义链的长度比可以是19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些实施方式中，所述siRNA正义链和反义链的长度比为19/21、21/23或23/25。
在一些实施方式中，本公开的具有稳定化修饰核苷酸的siRNA可以是以下第一种至第七种的siRNA，在下文中分别对每一种siRNA进行说明。
第一种siRNA
在一些实施方式中，本公开的siRNA是第一种siRNA。其中，
所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GAAUAAUGCUGGGACAGUZa1-3'(SEQ ID NO:1)；
5'-Za2ACUGUCCCAGCAUUAUUC-3'(SEQ ID NO:2),
其中，所述Za1为A,Za2为U，
并且，所述核苷酸序列I中包含位置对应于Za1的核苷酸Za3，所述核苷酸序列II中包含位置对应于Za2的核苷酸Za4，所述Za4是所述反义链5'末端的第一个核苷酸。在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:1所示的核苷酸序列。在本公开的上文与下文中，“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'端第一个核苷酸是位置对应于SEQ ID NO:1的第1个核苷酸的核苷酸。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间的差异包括Za4位置处的差异，且Za4选自A、G或C。在一些实施方式中，所述Za3是与Za4互补的核苷酸。在一些实施方式中，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间的差异为Za4位置处的差异，且Z4选自A、G或C。在一些实施方式中，本公开siRNA的核苷酸序列中的每个U或者T可任意地相互替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全反向互补。
在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反 向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，本公开的siRNA对目标基因表达的抑制活性得以进一步提升。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸为A；按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸选自A、G或C。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，所述核苷酸序列III和核苷酸序列IV中的每个核苷酸各自独立地为非氟代修饰的核苷酸的一种并且不是所述稳定化修饰核苷酸，所述核苷酸序列III和核苷酸序列IV的长度各自为1-4个核苷酸，所述核苷酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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。并且，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完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA中与前述第一段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A，所述第二段核苷酸序列的碱基为U；此时，正义链和反义链的长度比为20/20；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为GU，所述核苷酸序列IV的碱基组成为AC，所述第二段核苷酸序列的碱基组成为GU；此时，正义链和反义链的长度比为21/21；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为GGU，所述核苷酸序列IV的碱基组成为ACC，所述第二段核苷酸序列的碱基组成为GGU；此时，正义链和反义链的长度比为22/22；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为UGGU，所述核苷酸序列IV的碱基组成为ACCA，所述第二段核苷酸序列的碱基组成为UGGU；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补， 因此，给出了核苷酸序列III的碱基组成，核苷酸碱基IV的碱基组成也就确定了。
第二种siRNA
在一些实施方式中，本公开的siRNA是第二种siRNA，其中所述核苷酸序列I与SEQ ID NO:27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:28所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GCACCAAGGAUGAAGAGAZb1-3'(SEQ ID NO:27)；
5'-Zb2UCUCUUCAUCCUUGGUGC-3'(SEQ ID NO:28),
其中Zb1为U,Zb2为A，所述核苷酸序列I中包含位置对应于Zb1的核苷酸Zb3，所述核苷酸序列II中包含位置对应于Zb2的核苷酸Zb4，所述Zb4是所述反义链5'末端的第一个核苷酸。
在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:27所示的核苷酸序列。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:27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:28所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:28所示的核苷酸序列之间的差异包括Zb4位置处的差异，且Zb4选自U、G或C。在一些实施方式中，所述核苷酸差异为Zb4位置处的差异，且Zb4选自U、G或C。在一些实施方式中，Zb3是与Zb4互补的核苷酸。在一些实施方式中，本公开siRNA的核苷酸序列中的每个U或者T可任意地相互替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全反向互补。在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，本公开的siRNA对目标基因表达的抑制活性得以进一步提升。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸为U；按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸选自U、G或C。
在一些实施方式中，所述正义链还含有核苷酸序列III,所述反义链还含有核苷酸序列IV，所述核苷酸序列III和核苷酸序列IV的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是所述稳定化修饰核苷酸，所述核苷酸序列III和核苷酸序列IV的长度各自为1-4个核苷酸，所述核苷酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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。并且，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完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA中与前述第一段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为A，所述核苷酸序列IV的碱基为U，所述第二段核苷酸序列的碱基为A；此时，正义链和反义链的长度比为20/20；或者，所述核苷酸序列III和核苷酸序列IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为CA，所述核苷酸序列IV的碱基组成为UG，所述第二段核苷酸序列的碱基组成为CA；此时，正义链和反义链的长度比为21/21；或者，所述核苷酸序列III和核苷酸 序列IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为UCA，所述核苷酸序列IV的碱基组成为UGA，所述第二段核苷酸序列的碱基组成为UCA，此时，正义链和反义链的长度比为22/22；或者，所述核苷酸序列III和核苷酸序列IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为CUCA，所述核苷酸序列IV的碱基组成为UGAG，所述第二段核苷酸序列的碱基组成为CUCA；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基组成，核苷酸序列IV的碱基组成也就确定了。
第三种siRNA
在一些实施方式中，本公开的siRNA是第三种siRNA。其中，所述核苷酸序列I与SEQ ID NO:53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:54所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-CACCAAGGAUGAAGAGAUZc1-3'(SEQ ID NO:53)；
5'-Zc2AUCUCUUCAUCCUUGGUG-3'(SEQ ID NO:54),
其中Zc1为U,Zc2为A，所述核苷酸序列I中包含位置对应于Zc1的核苷酸Zc3，所述核苷酸序列II中包含位置对应于Zc2的核苷酸Zc4，所述Zc4是所述反义链5'末端的第一个核苷酸。
在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:53所示的核苷酸序列。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:53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:54所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:54所示的核苷酸序列之间的差异包括Zc4位置处的差异，且Zc4选自U、G或C。在 一些实施方式中，所述Zc3是与Zc4互补的核苷酸。在一些实施方式中，所述核苷酸序列II与SEQ ID NO:54所示的核苷酸序列之间的差异为Zc4位置处的差异，且Zc4选自U、G或C。在一些实施方式中，本公开siRNA的核苷酸序列中的每个U或者T可任意地相互替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全反向互补。在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，本公开的siRNA对目标基因表达的抑制活性得以进一步提升，同时脱靶效应出人意料地进一步降低。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸为A；按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸选自A、G或C。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，所述核苷酸序列III和核苷酸序列IV中的每个核苷酸各自独立地为非氟代修饰的核苷酸的一种并且不是所述稳定化修饰核苷酸，所述核苷酸序列III和核苷酸序列IV的长度各自为1-4个核苷酸，所述核苷酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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。并且，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完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA 中与前述第一段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为G，核苷酸序列IV的碱基为C，所述第二段核苷酸序列的碱基为G；此时，正义链和反义链的长度比为20/20；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为AG，所述核苷酸序列IV的碱基组成为CU，所述第二段核苷酸序列的碱基组成为AG；此时，正义链和反义链的长度比为21/21；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为CAG，所述核苷酸序列IV的碱基组成为CUG，所述第二段核苷酸序列的碱基组成为CAG；此时，正义链和反义链的长度比为22/22；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为UCAG，所述核苷酸序列IV的碱基组成为CUGA，所述第二段核苷酸序列的碱基组成为UCAG；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基组成，核苷酸碱基IV的碱基组成也就确定了。
第四种siRNA
在一些实施方式中，本公开的siRNA是第四种siRNA。其中，所述核苷酸序列I与SEQ ID NO:79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:80所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-UCUGAUAGAUGGAAUACUZd1-3'(SEQ ID NO:79)；
5'-Zd2AGUAUUCCAUCUAUCAGA-3'(SEQ ID NO:80),
其中Zd1为U,Zd2为A，所述核苷酸序列I中包含位置对应于Zd1的核苷酸Zd3，所述核苷酸序列II中包含位置对应于Zd2的核苷酸Zd4，所述Zd4是所述反义链5'末端的第一个核苷酸。
在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:79所示的核苷酸序列。在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:79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:80所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:80所示的核苷酸序列之间的差异包括Zd4位置处的差异，且Zd4选自U、G或C。在一些实施方式中，所述Zd3是与Zd4互补的核苷酸。在一些实施方式中，所述核苷酸序列II与SEQ ID NO:80所示的核苷酸序列之间的差异为Zd4位置处的差异，且Zd4选自U、G或C。在一些实施方式中，本公开siRNA的核苷酸序列中的每个U或者T可任意地相互替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全反向互补。在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，本公开的siRNA对目标基因表达的抑制活性得以进一步提升。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸为A；按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸选自A、G或C。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，所述核苷酸序列III和核苷酸序列IV中的每个核苷酸各自独立地为非氟代修饰的核苷酸的一种并且不是所述稳定化修饰核苷酸，所述核苷酸序列III和核苷酸序列IV的长度各自为1-4个核苷酸，所述核苷 酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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。并且，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完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA中与前述第一段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为G，核苷酸序列IV的碱基为C，所述第二段核苷酸序列的碱基为G；此时，正义链和反义链的长度比为20/20；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为AG，所述核苷酸序列IV的碱基组成为CU，所述第二段核苷酸序列的碱基组成为AG；此时，正义链和反义链的长度比为21/21；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为AAG，所述核苷酸序列IV的碱基组成为CUU，所述第二段核苷酸序列的碱基组成为AAG；此时，正义链和反义链的长度比为22/22；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为GAAG，所述核苷酸序列IV的碱基组成为CUUC，所述第二段核苷酸序列的碱基组成为GAAG；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基组成，核苷酸碱基IV的碱基组成也就确定了。
第五种siRNA
在一些实施方式中，本公开的siRNA是第五种siRNA。其中，所述核苷酸序列I与SEQ ID NO:105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:106所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-CUGAUAGAUGGAAUACUUZe1-3'(SEQ ID NO:105)；
5'-Ze2AAGUAUUCCAUCUAUCAG-3'(SEQ ID NO:106),
其中Ze1为A,Ze2为U，所述核苷酸序列I中包含位置对应于Ze1的 核苷酸Ze3，所述核苷酸序列II中包含位置对应于Ze2的核苷酸Ze4，所述Ze4是所述反义链5'末端的第一个核苷酸。
在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:105所示的核苷酸序列。在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:105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:106所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:106所示的核苷酸序列之间的差异Ze4位置处的差异，且Ze4选自A、G或C。在一些实施方式中，所述Ze3是与Ze4互补的核苷酸。在一些实施方式中，所述核苷酸序列II与SEQ ID NO:106所示的核苷酸序列之间的差异为Ze4位置处的差异，且Ze4选自A、G或C。在一些实施方式中，本公开siRNA的核苷酸序列中的每个U或者T可任意地相互替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全反向互补。在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，本公开的siRNA对目标基因表达的抑制活性得以进一步提升。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸为A；按 照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸选自A、G或C。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，所述核苷酸序列III和核苷酸序列IV中的每个核苷酸各自独立地为非氟代修饰的核苷酸的一种并且不是所述稳定化修饰核苷酸，所述核苷酸序列III和核苷酸序列IV的长度各自为1-4个核苷酸，所述核苷酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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。并且，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完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA中与前述第一段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A，所述第二段核苷酸序列的碱基为U；此时，正义链和反义链的长度比为20/20；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为GU，所述核苷酸序列IV的碱基组成为AC，所述第二段核苷酸序列的碱基组成为GU；此时，正义链和反义链的长度比为21/21；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为AGU，所述核苷酸序列IV的碱基组成为ACU，所述第二段核苷酸序列的碱基组成为AGU；此时，正义链和反义链的长度比为22/22；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为AAGU，所述核苷酸序列IV的碱基组成为ACUU，所述第二段核苷酸序列的碱基组成为AAGU；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基组成，核苷酸碱基IV的碱基组成也就确定了。
第六种siRNA
在一些实施方式中，本公开的siRNA是第六种siRNA。其中，所述 核苷酸序列I与SEQ ID NO:131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:132所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GAUGGAAUACUUACCAAUZf1-3'(SEQ ID NO:131)；
5'-Zf2AUUGGUAAGUAUUCCAUC-3'(SEQ ID NO:132),
其中Zf1为A,Zf2为U，所述核苷酸序列I中包含位置对应于Zf1的核苷酸Zf3，所述核苷酸序列II中包含位置对应于Zf2的核苷酸Zf4，所述Zf4是所述反义链5'末端的第一个核苷酸。
在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:131所示的核苷酸序列。在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:131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:132所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:132所示的核苷酸序列之间的差异Zf4位置处的差异，且Zf4选自A、G或C。在一些实施方式中，所述Zf3是与Zf4互补的核苷酸。在一些实施方式中，所述核苷酸序列II与SEQ ID NO:132所示的核苷酸序列之间的差异为Zf4位置处的差异，且Zf4选自A、G或C。在一些实施方式中，本公开siRNA的核苷酸序列中的每个U或者T可任意地相互替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全 反向互补。在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，本公开的siRNA对目标基因表达的抑制活性得以进一步提升。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸为A；按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸选自A、G或C。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，所述核苷酸序列III和核苷酸序列IV中的每个核苷酸各自独立地为非氟代修饰的核苷酸的一种并且不是所述稳定化修饰核苷酸，所述核苷酸序列III和核苷酸序列IV的长度各自为1-4个核苷酸，所述核苷酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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。并且，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完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA中与前述第一段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为A，核苷酸序列IV的碱基为U，所述第二段核苷酸序列的碱基为A；此时，正义链和反义链的长度比为20/20；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为UA，所述核苷酸序列IV的碱基组成为UA，所述第二段核苷酸序列的碱基组成为UA；此时，正义链和反义链的长度比为21/21；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为AUA，所述核苷酸序列IV的碱基组成为UAU，所述第二段核苷酸序列的碱基组成为AUA；此时，正义链和反义链的长度比为22/22；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为GAUA，所述核苷酸序列IV的碱基组成为UAUC，所述第二段核苷 酸序列的碱基组成为GAUA；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基组成，核苷酸碱基IV的碱基组成也就确定了。
第七种siRNA
在一些实施方式中，本公开的siRNA是第七种siRNA。其中，所述核苷酸序列I与SEQ ID NO:157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:158所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GAAUACUUACCAAUAAGAZg1-3'(SEQ ID NO:157)；
5'-Zg2UCUUAUUGGUAAGUAUUC-3'(SEQ ID NO:158),
其中Zg1为A,Zg2为U，所述核苷酸序列I中包含位置对应于Zg1的核苷酸Zg3，所述核苷酸序列II中包含位置对应于Zg2的核苷酸Zg4，所述Zg4是所述反义链5'末端的第一个核苷酸。
在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:157所示的核苷酸序列。在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:157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:158所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:158所示的核苷酸序列之间的差异Zg4位置处的差异，且Zg4选自A、G或C。在一些实施方式中，所述Zg3是与Zg4互补的核苷酸。在一些实施方式中，所述核苷酸序列II与SEQ ID NO:158所示的核苷酸序列之间的差异为Zg4位置处的差异，且Zg4选自A、G或C。在一些实施方式中，本公开siRNA的核苷酸序列中的每个U或者T可任意地相互替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上 反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全反向互补。在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，本公开的siRNA对目标基因表达的抑制活性得以进一步提升。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸为U；按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸选自U、G或C。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，所述核苷酸序列III和核苷酸序列IV中的每个核苷酸各自独立地为非氟代修饰的核苷酸的一种并且不是所述稳定化修饰核苷酸，所述核苷酸序列III和核苷酸序列IV的长度各自为1-4个核苷酸，所述核苷酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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。并且，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完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA中与前述第一段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为G，核苷酸序列IV的碱基为C，所述第二段核苷酸序列的碱基为G；此时，正义链和反义链的长度比为20/20；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为UG，所述核苷酸序列IV的碱基组成为CA，所述第二段核苷酸序列 的碱基组成为UG；此时，正义链和反义链的长度比为21/21；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为AUG，所述核苷酸序列IV的碱基组成为CAU，所述第二段核苷酸序列的碱基组成为AUG；此时，正义链和反义链的长度比为22/22；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为GAUG，所述核苷酸序列IV的碱基组成为CAUC，所述第二段核苷酸序列的碱基组成为GAUG；此时，正义链和反义链的长度比为23/23。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基组成，核苷酸碱基IV的碱基组成也就确定了。
以下，对于核苷酸序列V、siRNA中的核苷酸修饰以及修饰序列的描述适用于上述本公开的siRNA，例如第一种siRNA、第二种siRNA、第三种siRNA、第四种siRNA、第五种siRNA、第六种siRNA和第七种siRNA。即如果没有特指，下面对siRNA的描述应视为对上述本公开的七种siRNA，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“本公开的siRNA，例如上述第一种siRNA、第二种siRNA、第三种siRNA、第四种siRNA、第五种siRNA、第六种siRNA或第七种siRNA还含有核苷酸序列V”。
在一些实施方式中，所述正义链和反义链长度不同，所述反义链还含有核苷酸序列V，核苷酸序列V的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是所述稳定化修饰核苷酸，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3'末端，构成反义链的3'突出端。由此，本公开提供的siRNA正义链和反义链的长度比可以是19/20、19/21、19/22、20/21、20/22、20/23、21/22、21/23、21/24、22/23、22/24、22/25、23/24、23/25或23/26。在一些实施方式中，所述核苷酸序列V的长度为2个核苷酸，由此，本公开提供的siRNA正义链和反义链的长度比可以是19/21、21/23或23/25。
所述核苷酸序列V中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约成本，在一些实施方式中，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)、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HSD mRNA的亲和力，靶核苷酸序列V与第三段核苷酸序列完全反向互补，所述第三段核苷酸序列是指HSD17B13基因表达的mRNA中与第一段核苷酸序列的5'末端或第二段核苷酸序列的5'末端相邻、且长度与所述核苷酸序列V相等的核苷酸序列。因此，在一些实施方式中，本公开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mRNA沉默活性。
在一些实施方式中，对于所述第一种siRNA，所述第一段核苷酸序列具有SEQ ID NO:1所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是GU；所述siRNA的正义链含有如SEQ ID NO:3所示的核苷酸序列，所述反义链含有如SEQ ID NO:4所示的核苷酸序列：
5'-GAACAGAGAUACUACGGUZa3-3'(SEQ ID NO:3)；
5'-Za4ACUGUCCCAGCAUUAUUCAC-3'(SEQ ID NO:4),
其中，所述Za4是反义链5'末端的第一个核苷酸，Za3选自A、U、G或C，并且Za3是与Za4互补的核苷酸。
或者，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'-GUGAAUAAUGCUGGGACAGUZa3-3'(SEQ ID NO:5)；
5'-Za4ACUGUCCCAGCAUUAUUCACCA-3'(SEQ ID NO:6),
其中，所述Za4是反义链5'末端的第一个核苷酸，Za3选自A、U、G或C，并且Za3是与Za4互补的核苷酸。
在一些实施方式中，对于所述第二种siRNA，所述第一段核苷酸序列具有SEQ ID NO:29所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是CA。
所述siRNA的正义链含有如SEQ ID NO:29所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:30所示的核苷酸序列：
5'-GCACCAAGGAUGAAGAGAZb3-3'(SEQ ID NO:29)；
5'-Zb4UCUCUUCAUCCUUGGUGCUG-3'(SEQ ID NO:30),
其中，所述Zb4是反义链5'末端的第一个核苷酸，Zb3选自A、U、G或C，并且Zb4是与Zb3互补的核苷酸；
所述siRNA的正义链含有如SEQ ID NO:31所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:32所示的核苷酸序列：
5'-CAGCACCAAGGAUGAAGAGAZb3-3'(SEQ ID NO:31)；
5'-Zb4UCUCUUCAUCCUUGGUGCUGAG-3'(SEQ ID NO:32),
其中，所述Zb4是反义链5'末端的第一个核苷酸，Zb3选自A、U、G或C，并且Zb4是与Zb3互补的核苷酸。
在一些实施方式中，对于所述第三种siRNA，所述第一段核苷酸序列具有SEQ ID NO:55所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是AG；所述siRNA的正义链含有如SEQ ID NO:55所示的核苷酸序列，所述反义链含有如SEQ ID NO:56所示的核苷酸序列：
5'-CACCAAGGAUGAAGAGAUZc3-3'(SEQ ID NO:55)；
5'-Zc4AUCUCUUCAUCCUUGGUGCU-3'(SEQ ID NO:56),
其中，所述Zc4是反义链5'末端的第一个核苷酸，Zc3选自A、U、G或C，并且Zc4是与Zc3互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:57所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:58所示的核苷酸序列：
5'-AGCACCAAGGAUGAAGAGAUZc3-3'(SEQ ID NO:57)；
5'-Zc4AUCUCUUCAUCCUUGGUGCUGA-3'(SEQ ID NO:58),
其中，所述Zc4是反义链5'末端的第一个核苷酸，Zc3选自A、U、G或C，并且Zc4是与Zc3互补的核苷酸。
在一些实施方式中，对于所述第四种siRNA，所述第一段核苷酸序列具有SEQ ID NO:81所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是AG；所述siRNA的正义链含有如SEQ ID NO:81所示的核苷酸序列，所述反义链含有如SEQ ID NO:82所示的核苷酸序列：
5'-UCUGAUAGAUGGAAUACUZd3-3'(SEQ ID NO:81)；
5'-Zd4AGUAUUCCAUCUAUCAGACU-3'(SEQ ID NO:82),
其中，所述Zd4是反义链5'末端的第一个核苷酸，Zd3选自A、U、G或C，并且Zd4是与Zd3互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:83所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:84所示的核苷酸序列：
5'-AGUCUGAUAGAUGGAAUACUZd3-3'(SEQ ID NO:83)；
5'-Zd4AGUAUUCCAUCUAUCAGACUUC-3'(SEQ ID NO:84),
其中，所述Zd4是反义链5'末端的第一个核苷酸，Zd3选自A、U、G 或C，并且Zd4是与Zd3互补的核苷酸。
在一些实施方式中，对于所述第五种siRNA，所述第一段核苷酸序列具有SEQ ID NO:107所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是GU；所述siRNA的正义链含有如SEQ ID NO:107所示的核苷酸序列，所述反义链含有如SEQ ID NO:108所示的核苷酸序列：
5'-CUGAUAGAUGGAAUACUUZe3-3'(SEQ ID NO:107)；
5'-Ze4AAGUAUUCCAUCUAUCAGAC-3'(SEQ ID NO:108),
其中，所述Ze4是反义链5'末端的第一个核苷酸，Ze3选自A、U、G或C，并且Ze4是与Ze3互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:109所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:110所示的核苷酸序列：
5'-GUCUGAUAGAUGGAAUACUUZe3-3'(SEQ ID NO:109)；
5'-Ze4AAGUAUUCCAUCUAUCAGACUU-3'(SEQ ID NO:110),
其中，所述Ze4是反义链5'末端的第一个核苷酸，Ze3选自A、U、G或C，并且Ze4是与Ze3互补的核苷酸。
在一些实施方式中，对于所述第六种siRNA，所述第一段核苷酸序列具有SEQ ID NO:133所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是UA；所述siRNA的正义链含有如SEQ ID NO:133所示的核苷酸序列，所述反义链含有如SEQ ID NO:134所示的核苷酸序列：
5'-GAUGGAAUACUUACCAAUZf3-3'(SEQ ID NO:133)；
5'-Zf4AUUGGUAAGUAUUCCAUCUA-3'(SEQ ID NO:134),
其中，所述Zf4是反义链5'末端的第一个核苷酸，Zf3选自A、U、G或C，并且Zf4是与Zf3互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:135所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:136所示的核苷酸序列：
5'-UAGAUGGAAUACUUACCAAUZf3-3'(SEQ ID NO:135)；
5'-Zf4AUUGGUAAGUAUUCCAUCUAUC-3'(SEQ ID NO:136),
其中，所述Zf4是反义链5'末端的第一个核苷酸，Zf3选自A、U、G或C，并且Zf4是与Zf3互补的核苷酸。
在一些实施方式中，对于所述第七种siRNA，所述第一段核苷酸序列具有SEQ ID NO:159所示的核苷酸序列，所述第三段核苷酸序列的碱 基组成是UG；所述siRNA的正义链含有如SEQ ID NO:159所示的核苷酸序列，所述反义链含有如SEQ ID NO:160所示的核苷酸序列：
5'-GAAUACUUACCAAUAAGAZg3-3'(SEQ ID NO:159)；
5'-Zg4UCUUAUUGGUAAGUAUUCCA-3'(SEQ ID NO:160),
其中，所述Zg4是反义链5'末端的第一个核苷酸，Zg3选自A、U、G或C，并且Zg4是与Zg3互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:161所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:162所示的核苷酸序列：
5'-UGGAAUACUUACCAAUAAGAZg3-3'(SEQ ID NO:161)；
5'-Zg4UCUUAUUGGUAAGUAUUCCAUC-3'(SEQ ID NO:162),
其中，所述Zg4是反义链5'末端的第一个核苷酸，Zg3选自A、U、G或C，并且Zg4是与Zg3互补的核苷酸。
如前所述，本公开的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸，在一些实施方式中，本公开的siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致本公开的siRNA抑制HSD17B13基因表达的功能明显削弱或丧失。
在本公开的上下文中，所使用的的术语“修饰的核苷酸”是指核苷酸的核糖基2'位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008,13(19-20):842-55中公开的修饰的核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列II的第2、6、14、16个核苷酸，如果不是所述稳定化修饰核苷酸的话，为氟代修饰的核苷酸。在一些实施方式中，所述核苷酸序列II中的全部核苷酸均为修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列II的第2、6、14、16个核苷酸，如果不是所述稳定化修饰核苷酸的话，为氟代修饰的核苷酸，所述核苷酸序列II中的其它核苷酸各自独立地为非氟代修饰的核苷酸的一种。在一些实施方式中，按照5'末端到3'末端 的方向，所述核苷酸序列I的第7-9个核苷酸为氟代修饰的核苷酸。在一些实施方式中，所述核苷酸序列I中的全部核苷酸均为修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列I的第7-9个核苷酸为氟代修饰的核苷酸，所述核苷酸序列I中的其它核苷酸各自独立地为非氟代修饰的核苷酸的一种。本公开的siRNA通过具有上述修饰，能够实现基因表达调节活性和体内稳定性的良好平衡。
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，其具有以下式(7)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸、或核苷酸类似物。
在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
这些核糖基2'位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2'-烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-脱氧核苷酸中的一种。
在一些实施方式中，2'-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2'-OMe)，如式(8)所示。在一些实施方式中，2'-氨基修饰的核苷酸(2'-NH2)如式(9)所示。在一些实施方式中，2'-脱氧核苷酸(DNA)如式(10)所示：
本领域技术人员知晓各种对核苷酸的碱基进行修饰的方式。在一些实施方式中，碱基修饰包括但不限于在碱基上增加一个或多个甲基。在一些实施方式中，将胸腺嘧啶(T)视为碱基经修饰的尿嘧啶(U)的一种。在一些实施方式中，将5-甲基胞嘧啶视为碱基经修饰的胞嘧啶(C) 的一种。
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3'-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2'-、4'-位处以提供一个2',4'-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12)所示，ENA如式(13)所示，cET BNA如式(14)所示：
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中，无环核苷酸可以是解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(15)所示，GNA如式(16)所示：
上述式(15)和式(16)中，R选自H、OH或烷氧基(O-烷基)。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方式中，异核苷酸可以是碱基从核糖环的1'-位移动至2'-位或3'-位而形成的化合物，如式(17)或(18)所示。
上述式(17)-式(18)化合物中，Base表示核酸碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”、“2'-氟修饰的核苷酸”、“核糖基团的2'-羟基被氟取代的核苷酸”和“具有2’-氟代核糖基的核苷酸”意义相同，均指核苷酸的2'-羟基被氟取代，而形成的具有如式(7)所示结构的化合物；“甲氧基修饰的核苷酸”、“2'-甲氧基修饰的核苷酸”、“核糖基团的2'-羟基被甲氧基取代的核苷酸”和“具有2’-甲氧基核糖基的核苷酸”意义相同，均指核苷酸核糖基团的2'-羟基被甲氧基取代而形成的具有如式(8)所示结构的化合物。
在一些实施方式中，其中，所述反义链中不多于3个非氟代修饰的核苷酸为2'-脱氧核苷酸，其余每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸；或者，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。
在一些实施方式中，本公开的包含稳定化修饰核苷酸的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，所述反义链中第3位或第5位的核苷酸为稳定化修饰核苷酸，第18位的核苷酸为2'-脱氧核苷 酸或甲氧基修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。
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，上述修饰并未显著降低siRNA的抑制性能，具有上述修饰的siRNA还具有低的脱靶效应。
在一些实施方式中，本公开提供的所述siRNA为为下表1a-1g中列出的siHSDa1-M1、siHSDa1-M2、siHSDa2-M1、siHSDa2-M2、siHSDb1-M1、siHSDb1-M2、siHSDb2-M1、siHSDb2-M2、HSDc1-M1、siHSDc1-M2、siHSDc2-M1、siHSDc2-M2、HSDd1-M1、siHSDd1-M2、siHSDd2-M1、siHSDd2-M2、siHSDe1-M1、siHSDe1-M2、siHSDe2-M1、siHSDe2-M2、siHSDf1-M1、siHSDf1-M2、siHSDf2-M1、siHSDf2-M2、siHSDg1-M1、siHSDg1-M2、siHSDg2-M1或siHSDg2-M2中的一种。。
在一些实施方式中，本公开提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分为具有修饰基团的磷酸酯基。在一些实施方式中，具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基；在一些实施方式中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯基：
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。
在一些实施方式中，本公开提供的siRNA中，硫代磷酸酯基连接存在于由以下位置组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯基连 接存在于除正义链5'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于除正义链3'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于以下位置中的至少一处：
所述正义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
所述正义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
所述正义链的3'末端端部第1个核苷酸和第2个核苷酸之间；
所述正义链的3'末端端部第2个核苷酸和第3个核苷酸之间；
所述反义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
所述反义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
所述反义链的3'末端端部第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'末端端部第2个核苷酸和第3个核苷酸之间。
在一些实施方式中，本公开的siRNA为表1a-1g中列出的siHSDa1-M1S、siHSDa1-M2S、siHSDa2-M1S、siHSDa2-M2S、siHSDb1-M1S、siHSDb1-M2S、siHSDb2-M1S、siHSDb2-M2S、HSDc1-M1S、siHSDc1-M2S、siHSDc2-M1S、siHSDc2-M2S、HSDd1-M1S、siHSDd1-M2S、siHSDd2-M1S、siHSDd2-M2S、siHSDe1-M1S、siHSDe1-M2S、siHSDe2-M1S、siHSDe2-M2S、siHSDf1-M1S、siHSDf1-M2S、siHSDf2-M1S、siHSDf2-M2S、siHSDg1-M1S、siHSDg1-M2S、siHSDg2-M1S或siHSDg2-M2S中的一种。在一些实施方式中，所述siRNA反义链的5'末端核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。
常用的所述5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5'-磷酸核苷酸可具有如下结构：
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：
其中，R选自H、OH、甲氧基、氟；Base表示核酸碱基，选自A、U、C、G或T。
在一些实施方式中，5'-磷酸核苷酸为式(2)所示的含有5'-磷酸修饰的核苷酸，5'-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者为硫代磷酸酯修饰的核苷酸，如式(5)所示。
在一些实施方式中，本公开的siRNA为下表1a-1g中列出的siHSDa1-M1P、siHSDa1-M2P、siHSDa2-M1P、siHSDa2-M2P、siHSDb1-M1P、siHSDb1-M2P、siHSDb2-M1P、siHSDb2-M2P、HSDc1-M1P、siHSDc1-M2P、siHSDc2-M1P、siHSDc2-M2P、HSDd1-M1P、siHSDd1-M2P、siHSDd2-M1P、siHSDd2-M2P、siHSDe1-M1P、siHSDe1-M2P、siHSDe2-M1P、siHSDe2-M2P、siHSDf1-M1P、siHSDf1-M2P、siHSDf2-M1P、siHSDf2-M2P、siHSDg1-M1P、siHSDg1-M2P、siHSDg2-M1P或siHSDg2-M2P中的一种。本公开的发明人意外发现，这些本公开提供的siRNA不仅具有显著增强的血浆和溶酶体稳定性，还保留很高的HSD mRNA抑制活性，并且还具有低的脱靶效应。
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Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
在一些实施方式中，所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，在一些实施方式中，siRNA与药学上可接受的载体的重量比可以为1:(1-500)，在一些的实施方式中，上述重量比为1:(1-50)。
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。
所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的 含量。在一些实施方式中，所述药物组合物所制成的制剂在给药过程中的剂量会因给药方式的不同而发生调整。
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺部、或通过喷雾经肺部给药到其它脏器组织(如肝脏)、或通过口服等方式递送所述药物组合物。在一些实施方式中，所述药物组合物通过皮下注射的方式给药。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于中国专利申请CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
在一些实施方式中，所述有机胺可为中国专利申请CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
其中：
每个X101或X102各自独立地是O、S、N-A或C-A，其中A是氢或C1-C20烃链；
每个Y101或Z101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO2；
每个R101、R102、R103、R104、R105、R106或R107各自独立地是氢，环 状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R102是氢；
并且，如果n或m中的至少一个是2，那么R103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。
在一些实施方式中，R103是多胺。在其它实施方式中，R103是缩酮。在一些实施方式中，在式(201)中的R101和R102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R103可以是下述式(204)-式(213)中的任一个：

其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
其中，式(201)所示化合物可以根据中国专利申请CN103380113A中的描述制备。
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
所述聚乙二醇化脂质为1,2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80):(19.7-80):(0.3-50)，例如可以为(50-70):(20-40):(3-20)。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成 的药物组合物中，siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1:1至约1:50、从约1:1至约1:30、从约1:3至约1:20、从约1:4至约1:18、从约1:5至约1:17、从约1:5至约1:15、从约1:5至约1:12、从约1:6至约1:12或从约1:6至约1:10的范围内，例如，本公开的siRNA与全部脂质的重量比为约1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17或1:18。
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的siRNA与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的siRNA替代现有siRNA即可；在一些实施方式中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的siRNA溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1：(2-5)，例如可以为1:4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％， 粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
siRNA缀合物
在另一方面，本公开提供了一种siRNA缀合物，所述siRNA缀合物含有本公开提供的siRNA，以及缀合连接至该siRNA的缀合基团。在一些实施方式中，所述缀合基团包含接头和药学上可接受的靶向基团和/或递送辅助基团，并且，所述siRNA、所述接头和所述靶向基团或者所述递送辅助基团依次共价或非共价连接，每个所述靶向基团选自能够和细胞表面受体结合的配体，每个递送辅助基团选自能够增加所述siRNA缀合物在递送目标器官或组织中的生物相容性的基团。
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。siRNA缀合物应根据上下文，理解为多个siRNA缀合物的总称或者某个化学式所表示的siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开的siRNA缀合物的特定化合物。
一般来说，所述缀合基团包含药学上可接受的至少一个靶向基团，或者进一步还包含接头(linker)，并且，所述siRNA、所述接头和所述靶向基团依次连接。在一些实施方式中，所述靶向基团为1-6个。在一些实施方式中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在siRNA正义链的3'端或5'端，也可在反义 链的5'端，还可以在siRNA的内部序列中。在一些实施方式中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3'末端。
在一些实施方式中，所述缀合基团可以连接在核苷酸的磷酸基团、2'-位羟基或者碱基上。在一些实施方式中，所述缀合基团还可以连接在3'-位羟基上，此时核苷酸之间采用2'-5'磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology,2015,10(5):1181-7.
靶向基团可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些接头、靶向基团的种类以及与siRNA的连接方式可参见WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。在一些实施方式中，所述siRNA与缀合基团间可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。
在一些实施方式中，所述靶向基团可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，至少一个或每个所述靶向基团选自能够和表达所述HSD17B13基因的细胞表面受体结合的配体。
在一些实施方式中，至少一个或每个所述靶向基团选自能够和哺乳动物肝实质细胞表面受体(ASGPR)结合的配体。在一些实施方式中，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。在一些实施方式中，每个所述靶向基团独立地为去唾液 酸糖蛋白或糖。在一些实施方式中，每个所述靶向基团独立地为去唾液酸糖蛋白，例如去唾液酸血清类枯蛋白(asialoorosomucoid,ASOR)或去唾液酸始球蛋白(asialofetuin，ASF)。在一些实施方式中，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。在一些实施方式中，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。在一些实施方式中，本公开的siRNA缀合物中的接头具有如式(301)所示的结构：
其中，k为1-3的整数；
LA具有如式(302)所示的包含酰胺键的结构，LB具有如式(303)所示的包含N-酰基吡咯烷的结构，含有羰基和氧原子，LC为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团；

其中，n302、q302和p302各自独立地为2-6的整数，可选地，n302、q302和p302各自独立地为2或3；n303为4-16的整数，可选地，n303为8-12的整数，表示基团共价连接的位点。
所述接头中，每个LA分别与一个所述靶向基团通过醚键连接，并通过LC部分中羟基的氧原子与LC部分形成醚键而连接；LB通过式(303)中的羰基与LC部分中氨基的氮原子形成酰胺键而连接，并通过式(303)中的氧原子与所述siRNA通过氧原子形成磷酸酯键或硫代磷酸酯键相连接。
在一些实施方式中，本公开提供的siRNA缀合物具有如式(305)所示的结构：
其中，Nu表示本公开提供的siRNA。
在一些实施方式中，本公开的siRNA缀合物中的接头具有式(306)所示的结构：
其中，n306为0-3的整数，每个p306独立地为1-6的整数，表示基团共价连接的位点；所述连接基团通过由*标出的氧原子与所述靶向基团形成醚键连接；所述连接基团由#标出的氧原子中的至少一个与所述siRNA形成磷酸酯键或硫代磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C1-C3烷基连接形成C1-C3烷氧基；
在一些实施方式中，本公开的siRNA缀合物具有如式(307)所示的结构：
其中，Nu表示本公开提供的siRNA。
在一些实施方式中，本公开的siRNA缀合物具有式(308)所示的结构：
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；
R10、R11、R12、R13、R14或R15各自独立地为H，或选自于由以下基团所组成的组：C1-C10烷基、C1-C10卤代烷基以及C1-C10烷氧基；
R3具有式A59所示的结构：
其中，E1为OH、SH或BH2，Nu表示本公开提供的siRNA；
R2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、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中R2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：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基苯基)、-CONH(C1-C10烷基苯基)、-CONH2、-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、 -C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
每个L1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可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、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，L1可选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
表示基团共价连接的位点；
M1表示靶向基团，其定义和可选择的范围与上述相同。在一些实施方式中，每个M1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。
技术人员会理解的是，尽管为了方便起见，L1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/或置换而产生的胺或烯基。为了本公开内容的目的，L1的长度是连接两个附着点的链中的原子数。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。
当M1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体时，在一些实施方式中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述缀合物中M1配体的个数至少为2；在一些实施方式中，n1+n3≥2，这样可以使得M1配体的个数至少为3，使得M1配体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M1配体的个数大于3个时，M1配体与肝表面去唾液酸糖蛋白受体结合的容易程度增加并不明显，因此，从合成容易程度、结构/工艺成本和递送效率等多方面综合考虑，在一些实施方式中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。
在一些实施方式中，m1、m2和m3独立地选自2-10的整数时，可以使多个M1配体之间的空间位置适合M1配体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缀合物更为简单，更容易合成和/或降低成本，在一些实施方式中，m1、m2和m3各自独立地为2-5的整数，在一些实施方式中，m1＝m2＝m3。
本领域技术人员可以理解，当R10、R11、R12、R13、R14和R15各自独立地选自H、C1-C10烷基、C1-C10卤代烷基、以及C1-C10烷氧基中的一种时，不会改变本文公开的缀合物的性质，均可以实现本公开的目的。在一些实施方式中，R10、R11、R12、R13、R14和R15各自独立地选自H、甲基和乙基。在一些实施方式中，R10、R11、R12、R13、R14和R15均为H。
根据本公开提供的siRNA缀合物，R3为式A59所示结构的基团，其中，E1为OH、SH或BH2，基于制备原料易获取性的考虑，在一些实施方式中，E1为OH或SH。
在一些实施方式中，R2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R10、R11、R12、R13、R14和R15的碳原子与N原子互相连接的链状结构。因此，R2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N原子的连接基团。在一些实施方式中，在通过固相合成的工艺制备本公开的siRNA缀合物的情况下，R2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R3中的P原子相连接的连接位点。在一些实施方式中，R2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 R3上的P原子连接的位点与P原子形成磷酸酯键。在一些实施方式中，R2的长度为2-20个原子、或者4-15个原子。在一些实施方式中，R2是B5、B6、B5’或B6’：
其中，表示基团共价键连接的位点。
q2的取值范围可以是1-10的整数，在一些实施方式中，q2为1-5的整数。
L1的作用是将M1配体与含氮骨架上的N连接，为本公开的siRNA缀合物提供靶向功能。在一些实施方式中，L1选自式A1-A26基团中的一种或多种的连接组合。在一些实施方式中，L1选自A1、A4、A5、A6、A8、A10、A11和A13中的一种或多种的连接组合；在一些实施方式中，L1选自A1、A4、A8、A10和A11中至少2个的连接组合；在一些实施方式中，L1选自A1、A8、A10中至少2个的连接组合。

在一些实施方式中，L1的长度可以为3-25个原子，3-20个原子、4-15个原子或5-12个原子。在一些实施方式中是，L1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。
在一些实施方式中，j1为2-10的整数，在一些实施方式中，j1为3-5的整数。在一些实施方式中，j2为2-10的整数，在一些实施方式中，j2为3-5的整数。R’为C1-C4的烷基，在一些实施方式中，R’为甲基、乙基和异丙基中的一种。Ra为A27、A28、A29、A30和A31中的一种， 在一些实施方式中，Ra为A27或A28。Rb为C1-C5的烷基，在一些实施方式中，Rb为甲基、乙基、异丙基和丁基中的一种。在一些实施方式中，在式A1-A26中各自对j1、j2、R’、Ra、Rb进行选择，以实现M1配体与含氮骨架上的N连接，并使M1配体之间的空间位置更适合M1配体与肝表面去唾液酸糖蛋白受体结合。
在一些实施方式中，本公开的siRNA缀合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：






其中，Nu表示本公开的siRNA。
在一些实施方式中，式A59中的P原子可以连接到siRNA序列中任何可能的位置，例如，式A59中的P原子可以连接到siRNA正义链或反义链的任何一个核苷酸上；在一些实施方式中，式A59中的P原子连接到siRNA正义链的任何一个核苷酸上。在一些实施方式中，式A59中的P原子连接到siRNA正义链或反义链的端部；在一些实施方式中，式A59中的P原子连接到siRNA正义链的端部。所述端部指所述正义链或所述反义链中从其一端起算的前4个核苷酸。在一些实施方式中，式A59中的P原子连接到siRNA正义链或反义链的末端；在一些实施方式中，式A59中的P原子连接到siRNA正义链的3'末端。在连接至siRNA的正义链的上述位置的情况下，本公开提供的缀合物进入细胞后，在解旋时，可以释放出单独的siRNA反义链，以通过RNAi机制抑制靶基因表达。
式A59中的P原子可以连接到siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5'位、核苷酸的2'位、核苷酸的3'位或核苷酸的碱基上。在一些实施方式中，式A59中的P原子可通过形成磷酸二酯键连接至所述siRNA中的核苷酸的2'位、3'位或5'位。在一些实施方式中，式A59中的P原子连接在siRNA正义链3'末端核苷酸的3'羟基脱氢后形成的氧原子上，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。
本公开的发明人发现，本公开的siRNA以及含有这些siRNA的siRNA缀合物表现出具有显著提高的血浆中稳定性和低脱靶效应的同时，还表现出较高的HSD17B13沉默活性。因此，在一些实施方式中，本公开的siRNA可以为表1a和1b中示出的siRNA中的一种。
表1a本公开的第一种siRNA序列

表1b本公开的第二种siRNA序列

表1c本公开的第三种siRNA序列

表1d本公开的第四种siRNA序列
表1e本公开的第五种siRNA序列
表1f本公开的第六种siRNA序列

表1g本公开的第七种siRNA序列

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d表示该字母d右侧相邻的一个核苷酸为2'-脱氧核苷酸；下划线标出的大写字母S表示该字母S左侧相邻的一个核苷酸为稳定化修饰核苷酸；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；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。在一些实施方式中，S是表示具体的稳定化修饰例如moe，其中，下划线标出的字母组合moe表示在该字母组合moe左侧相邻的一个核苷酸为具有2'-O-甲氧基乙基修饰的核苷酸。在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。另外，上述表1a-1g中所列的序列中的每个U或者T可任意地相互替换，不会对siRNA的活性或脱靶效应产生明显影响。
本公开siRNA缀合物的制备
上述siRNA缀合物可以通过现有技术中已经详细描述的方法进行合 成。例如，WO2015006740A2中详细描述了多种siRNA缀合物的制备方法。也可以通过本领域技术人员熟知的方式，获得本公开的siRNA缀合物。如WO2014025805A1中记载了式(305)所示结构的制备方法，Rajeev等人在ChemBioChem 2015,16,903-908中描述了式(307)所示结构的制备方法。中国专利申请CN110959011A也详细公开了制备式(308)所示的siRNA缀合物的方法。以引用的方式将上述文献内容整体并入本文。
本公开的siRNA缀合物也可以与药学上可接受的其它辅料联用，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种，详情可参见上文关于本公开的药物组合物的描述。
本公开的siRNA、药物组合物及siRNA缀合物的应用
在一些实施方式中，本公开提供了本公开的siRNA，和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防与HSD17B13基因表达的mRNA水平相关的疾病或症状的药物中的用途。在一些实施方式中，本公开提供了本公开的siRNA，和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防慢性纤维炎性肝病的药物中的用途。
在一些实施方式中，本公开提供了一种治疗和/或预防与HSD17B13基因表达的mRNA水平相关的疾病或症状的方法，所述方法包括向有需要的受试者给予本公开的siRNA，和/或本公开的药物组合物和/或本公开的siRNA缀合物。在一些实施方式中，所述与HSD17B13基因表达的mRNA水平相关的疾病或症状是慢性纤维炎性肝病。在一些实施方式中，所述慢性纤维炎性肝病选自由下列所组成的组：肝炎、肝纤维化、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪性肝病(NAFLD)、肝硬化、酒精性脂肪性肝炎(ASH)、酒精性脂肪肝病(ALD)、丙型肝炎(HCV)相关的硬化、药物引起的肝损伤、及肝细胞坏死。
在一些实施方式中，本公开还提供了一种抑制细胞中HSD17B13基因水平表达的方法，所述方法包括将有效剂量的本公开的siRNA，和/或药物组合物和/或siRNA缀合物与所述细胞接触。
通过将本公开提供的siRNA、药物组合物和/或siRNA缀合物给予有需要的受试者，可以通过对基因表达进行调控的机制达到预防和/或治疗 由细胞中HSD17B13基因的表达而引起的病理状况或疾病的目的。
因此，本公开提供的siRNA、药物组合物和/或siRNA缀合物可用于预防和/或治疗所述病理状况或疾病、或用于制备用于预防和/或治疗本文所述病理状况或疾病的药物。
本文所使用的术语“给药/给予”是指通过使得至少部分地将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整个身体相比将更多siRNA、药物组合物和/或siRNA缀合物递送至特定位点；而全身给药导致将所述siRNA、药物组合物和/或siRNA缀合物递送至受试者的基本整个身体。考虑到本公开旨在提供预防和/或治疗慢性纤维炎性肝病的手段，在一些实施方式中采用能够将药物递送至肝脏的给药方式。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月、每2个月、每3个月、每半年、或每年1次或多次。
本公开所述的siRNA、药物组合物和/或siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体死亡的致死量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中，指引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开所述的siRNA、药物组合物和/或siRNA缀合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或C3H/HeNCrlVr小鼠，以所述siRNA、药物组合物和/或siRNA缀合物中 的siRNA的量计：对于siRNA与药学上可接受的缀合分子形成的siRNA缀合物，其siRNA用量可以为0.001-100mg/kg体重，在一些实施方式中为0.01-50mg/kg体重，在进一步的实施方式中为0.05-20mg/kg体重，在更进一步的实施方式中为0.1-15mg/kg体重，在又进一步的实施方式中为0.1-10mg/kg体重。在给予本公开所述的siRNA、药物组合物和/或siRNA缀合物时，可优选上述用量。
另外，通过将本公开的siRNA、药物组合物和/或siRNA缀合物导入细胞，还可以通过RNAi的机制达到抑制细胞中HSD17B13基因的表达这一目的。
采用本公开提供的方法抑制HSD17B13基因在细胞中表达，所提供的siRNA、药物组合物和/或siRNA缀合物中的siRNA的用量是本领域技术人员根据期望获得的效果容易确定的。例如，在一些实施方式中，所提供的siRNA缀合物中的siRNA用量是这样的量：其足以减少靶基因的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或至约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、递送是局部还是全身等。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提供的siRNA、药物组合物和/或siRNA缀合物。
在一些实施方式中，本文所述的试剂盒可在一个容器中提供siRNA、药物组合物和/或siRNA缀合物。在一些实施方式中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方式中，本文所述的试剂盒可在不同于提供本文所述siRNA、药物组合物和/或siRNA缀合物的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将siRNA、药物组合物和/或siRNA缀合物与药学上可接受的载体和/或辅料或其它成分(若有的话) 进行混合的说明书。
在本公开的试剂盒中，所述siRNA和药学上可接受的载体和/或辅料以及所述药物组合物和/或siRNA缀合物，和/或药学上可接受的辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述siRNA和药学上可接受的载体和/或辅料以及所述药物组合物和/或siRNA缀合物和任选的药学上可接受的辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor Laboratory Press(1989))所记载的方法进行。
制备例1-7本公开提供的siRNA缀合物1-7的合成
按照CN110959011A制备例13所述的制备方法，制备获得了以下表2中的缀合物1-7，区别仅在于，各siRNA缀合物中含有的siRNA的正义链和反义链分别如表2中所示；按照以下表2中编号为缀合物1-缀合物7的siRNA的核酸序列，分别合成siRNA的正义链和反义链。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将各siRNA缀合物稀释至浓度为0.2mg/mL(以siRNA计)后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass SP1ectrometry，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，说明所合成的缀合物1-7是目标设计的双链核酸序列。各个siRNA缀合物分别具有式(403)所示的结构，并且该siRNA缀合物包含的siRNA分别具有表2中缀合物1-7所对应的siRNA序列。
其中，式(403)中的Nu为具有本公开表2中缀合物1-7所对应的siRNA序列的siRNA基团，并且缀合基团连接至siRNA基团正义链3'末端核苷酸的核糖3'位。对比制备例1-9参比siRNA缀合物的合成
按照CN110959011A制备例13所述的制备方法，制备获得了上文表2中编号为参比缀合物1、参比缀合物2、参比缀合物3、参比缀合物4、参比缀合物5、参比缀合物6、参比缀合物7、参比缀合物8和参比缀合物NC的参比siRNA缀合物，区别仅在于，各参比siRNA缀合物中含有的siRNA的正义链和反义链分别如表2中所示；按照以下表2中编号为参比缀合物1、参比缀合物2、参比缀合物3、参比缀合物4、参比缀合物5、参比缀合物6、参比缀合物7、参比缀合物8和参比缀合物NC的siRNA的核酸序列，分别合成siRNA的正义链和反义链。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将各参比siRNA缀合物稀释至浓度为0.2mg/mL(以siRNA计)后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。实测值与理论值一致，说明所合成的参比缀合物1、参比缀合物2、参比缀合物3、参比缀合物4、参比缀合物5、参比缀合物6、参比缀合物7、参比缀合物8和参比缀合物NC分别具有目标设计的双链核酸序列。各参比siRNA缀合物具有式(403)所示的结构，并且所包含的siRNA分别具有表2中参比缀合物1、参比缀合物2、参比缀合物4、参比缀合物5、参比缀合物6、参比缀合物7和参比缀合物NC所对应的siRNA序列。其中，参比缀合物1是和缀合物1相比具有基本相同的序列组成、但不包含稳定化修饰核苷酸的siRNA缀合物；参比缀合物2是和缀合物2相比具有基本相同的序列组成、但不包含稳 定化修饰核苷酸的siRNA缀合物；参比缀合物3是和缀合物3相比具有基本相同的序列组成、但不包含稳定化修饰核苷酸的siRNA缀合物；参比缀合物4是和缀合物4相比具有基本相同的序列组成、但不包含稳定化修饰核苷酸的siRNA缀合物；参比缀合物5是和缀合物5相比具有基本相同的序列组成、但不包含稳定化修饰核苷酸的siRNA缀合物；参比缀合物6是和缀合物6相比具有基本相同的序列组成、但不包含稳定化修饰核苷酸的siRNA缀合物；参比缀合物7是和缀合物7相比具有基本相同的序列组成、但不包含稳定化修饰核苷酸的siRNA缀合物；参比缀合物8是和缀合物9相比具有基本相同的序列组成、但不包含稳定化修饰核苷酸的siRNA缀合物；参比缀合物NC是序列与已知的mRNA均不具有显著同源性的阴性对照siRNA缀合物。
表2 siRNA缀合物中的siRNA序列


其中，大写字母C、G、U、A、T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；下划线标出的字母组合moe表示该字母组合moe左侧相邻的一个核苷酸为核糖2'-O-甲氧基乙基修饰的核苷酸；5mCmoe表示碱基为5-甲基胞嘧啶，且核糖被2'-O-甲氧基乙基修饰的核苷酸；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；小写字母d表示该字母d右侧相邻的一个核苷酸为2'-脱氧核苷酸，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为核糖环5’位具有(E)乙烯基磷酸基修饰的核苷酸，大写字母P表示该大写字母合VP右侧相邻的一个核苷酸为核糖环5’位具有磷酸酯基的核苷酸。
制备例8-12本公开提供的siRNA缀合物8-12的合成
按照CN110959011A制备例13所述的制备方法，制备获得了上表2中的缀合物8-10，区别仅在于，各siRNA缀合物中含有的siRNA的正义链和反义链分别如表2中所示；按照以上表2中编号为缀合物8-缀合物12的siRNA的核酸序列，分别合成siRNA的正义链和反义链。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将各siRNA缀合物稀释至浓度为0.2mg/mL(以siRNA计)后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass SP1ectrometry，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，说明所合成的缀合物8-12是目标设计的双链核酸序列。各个siRNA缀合物分别具有式(403)所示的结构，其中缀合基团共价连接至siRNA正义链3’末端核苷酸的核糖环3’位，并且该siRNA缀合物包含的siRNA基团分别具有表2中缀合物8-12所对应的siRNA序列。具体的分子量检测结果参见以下表2A：表2A siRNA缀合物分子量检测结果

制备例13本公开提供的siRNA缀合物Tm测定
称取上述制备的缀合物9或者参比缀合物8各25mg，分别用PBS配置成0.02mg/mL的供试品溶液。将供试品溶液置于石英比色(10mm)中。使用Agilent cary 3000 UV，设定波长260nm，按照说明书测定Tm值。测得缀合物9的Tm值为89.02℃，参比缀合物8的Tm值为88.07℃，表明与参比缀合物相比，本公开的具有基本相同的序列组成、但包含稳定化修饰核苷酸的siRNA的Tm增加0.95℃。
制备例14双链热解离温度Tm的测定
用1×PBS缓冲液将上述制备的缀合物11、缀合物12、参比缀合物10和参比缀合物11中的每一个分别配制为0.02mg/mL的溶液，作为供试品溶液。在配备有热式程序的Agilent cary300 UV分光光度计上的10mm路径长度石英比色皿中加入供试品溶液，在260nm波长下监测温度-吸光率曲线，其中加热速率为0.5℃/min，自20.0℃起始升温至95℃。双链热解离温度Tm按照分光光度计说明书由温度-吸光率曲线的一阶导数计算获得。Tm值和ΔTm值结果如以下表2B所示：
表2B双链热解离温度Tm
其中，对于缀合物11和参比缀合物10，
ΔTm值(待测缀合物)＝Tm(待测缀合物)-Tm(参比缀合物10)；
对于缀合物12和参比缀合物11，
ΔTm值(待测缀合物)＝Tm(待测缀合物)-Tm(参比缀合物11)。
根据表2B的结果可知，如说明书中所述，按照本公开的方式，在siRNA中引入稳定化修饰核苷酸，可以提高双链热解离温度，其Tm值升高。本公开提供的其他未给出Tm值的缀合物具有类似的性质。
实验例1 siRNA缀合物在体外psi-CHECK系统中对在靶目标序列的
抑制活性
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，将所述检测质粒与待测缀合物共转染至HEK293A细胞中，通过双萤光素酶报告基因的表达水平，来反映siRNA的目标序列抑制活性。具体步骤如下：
[1]构建检测质粒
采用psi-CHECKTM-2(PromegaTM)质粒构建了检测质粒，根据缀合物1、缀合物2和缀合物4分别构建检测质粒，所述质粒中分别含有目标序列1、目标序列2和目标序列3，即siRNA缀合物1靶序列为目标序列1，siRNA缀合物2靶序列为目标序列2，siRNA缀合物4靶序列为目标序列3。对于待测siRNA缀合物，目标序列如下所示：
目标序列1：
5’-TCGAGGTGAATAATGCTGGGACAGTAGC-3’
(SEQ ID NO:215)
目标序列2：
5’-TCGAGCAGCACCAAGGATGAAGAGATGC-3’
(SEQ ID NO:216)
目标序列3：
5’-TCGAGAGTCTGATAGATGGAATACTTGC-3’
(SEQ ID NO:217)
该目标序列1、目标序列2或目标序列3与HSD mRNA中的一部分同源，并与所检测的siRNA缀合物中反义链的序列完全互补，因此各siRNA缀合物对目标序列1的抑制效果可反映所检测的siRNA缀合物的目标基因表达的抑制能力。将目标序列1及其互补序列克隆到 psi-CHECKTM-2质粒的Xho I/Not I位点。
[2]转染
在添加有10％的胎牛血清(FBS，RMBIO公司)及0.2％体积比的青链霉素双抗(Penicillin-Streptomycin，HyClone公司)的H-DMEM完全培养基(HyClone公司)中，于37℃在含5％CO2/95％空气的培养箱中培养HEK293A细胞(购自南京科百生物技术有限公司)。
将HEK293A细胞以8×103细胞/孔接种于96孔板中，16小时后细胞生长密度达到70％时，吸尽培养孔中完全培养基，每孔加入80μL opti-MEM培养基(GIBCO公司)继续培养1.5小时。
用PBS将上述检测质粒稀释成20μM储存液；用PBS将每一种待测siRNA缀合物分别配制成20μM、4μM、1μM、0.25μM、0.0625μM、0.015625μM、0.003906μM、0.0009765μM、0.0002441μM、0.00006104μM和0.00001526μM(以siRNA缀合物中的siRNA量计)共11个不同浓度的siRNA缀合物工作液。所用siRNA缀合物分别为上述制备获得的缀合物1、缀合物2、缀合物4和参比缀合物1、参比缀合物2、参比缀合物4。
对于每一siRNA缀合物，分别配制1A1-1A11溶液，每份1A1-1A11溶液依次分别含有上述11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8.95μL的Opti-MEM培养基。
配制1B溶液，每份1B溶液含有0.2μL LipofectamineTM 2000和9.8μL 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8.95μL的Opti-MEM培养基。
分别将一份1B溶液与得到的一份每个siRNA缀合物的1A1-1A11溶液混合，分别室温下孵育20min，得到每个siRNA缀合物的转染复合物1X1-1X11。
将一份1B溶液与一份1C溶液混合，分别室温下孵育20min，得到空白转染复合物1Xa。
在培养孔中，分别加入每一siRNA缀合物的转染复合物1X1-1X11，均匀混合，加入量为20μL/孔，得到每个siRNA缀合物终浓度分别约为40nM、10nM、2.5nM、0.625nM、0.15625nM、0.03906nM、0.009765nM、0.002441nM、0.0006103nM、0.0001526nM、0.00003815nM(以siRNA缀合物中的siRNA量计)的转染复合物，每个siRNA缀合物的转染复合物1X1-1X11均转染3个培养孔，得到含siRNA缀合物的共转染混合物，记为测试组。
对于每一siRNA缀合物，在另外3个培养孔中，分别加入转染复合物1Xa，加入量为20μL/孔，得到不含siRNA缀合物的转染混合物，记为空白对照组。
分别将含siRNA缀合物的共转染混合物和不含siRNA缀合物的转染混合物在培养孔中转染4小时后，每孔补加100μL含20％FBS的H-DMEM完全培养基。将96孔板置于CO2培养箱在37℃、含5v/v％CO2的空气气氛下继续培养24小时。
[3]检测
吸去培养孔中的培养基，每孔加入150μL的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96孔酶标板上各培养孔中Firefly的化学发光值(Fir)；再向96孔酶标板上每孔加入60μLStop &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96孔酶标板上各培养孔中Renilla的化学发光值(Ren)。
计算96孔酶标板上每孔发光比值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报告基因的相对表达水平，即残留活性。siRNA对目标序列的抑制率＝(1-R)×100％。
依据转染了不同浓度的待测siRNA后，HEK293A细胞中Renilla的相对残留活性，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(four parameters)剂量-效应曲线。
根据拟合的剂量-效应曲线对应的函数，计算待测siRNA靶向目标序列的IC50值，所述函数如下，
式中：
Y是比值R，即Renilla的相对残留活性，
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'是当Y在底部到顶部之间一半时对应的X值，而HillSlope则是曲线在X'处的斜率。
由该剂量-效应曲线和对应的函数，确定当Y＝50％时对应的X50值，计算获得各siRNA的IC50值＝10^X50(nM)，IC50值总结于表3中。
表3 siRNA缀合物在psi-CHECK系统中的IC50
由表3的结果可知，本公开的siRNA缀合物在体外psi-CHECK系统中有很高的目标序列抑制活性。同时，本公开的siRNA缀合物与其序列相同、但不包含稳定化修饰核苷酸的参比缀合物1相比，参比缀合物1的IC50值是缀合物1的IC50的5倍以上，参比缀合物2的IC50值是缀合物2的IC50的3倍，缀合物4同样显示出极低的目标序列抑制的IC50值。这些结果表明，与参比缀合物相比，本公开的siRNA缀合物对目标序列具有显著提高的抑制活性。
实验例2 siRNA缀合物在猴原代肝细胞中对HSD mRNA表达量的抑制效率检测
在I型胶原蛋白包被的12孔培养板中，每孔中加入1000μL的细胞维持培养基(CM Culture medium，The Primary Cell Solution)，于37℃在含5％CO2/95％空气的培养箱中培养30min。
将冻存的猴原代肝细胞(The Primary Cell Solution)在含有10％胎牛血清(FBS，RMBIO公司)的解冻培养基(Thawing medium，The Primary Cell Solution公司)中，在37℃下解冻2min，在700rpm速度下离心5min，弃去上清液，以接种培养基(CM seeding medium，The Primary Cell Solution公司)溶解并使用细胞计数仪计数，随后加入接种培养基至活细胞量为4×105细胞/孔，于37℃在含5％CO2/95％空气的培养箱中培养过夜。
弃去培养基，每孔添加1000μL的细胞维持培养基(CM Culture medium，The Primary Cell Solution)，得到猴原代肝细胞悬液。
用DEPC水将siRNA缀合物6、缀合物7、参比缀合物6和参比缀合物7分别配制成20μM(以siRNA计)的siRNA缀合物工作液。
配制2A溶液，使用siRNA缀合物6配制2A溶液，每份2A溶液依次含有上述siRNA缀合物6工作液3μl和细胞维持培养基97μl。
配制2B溶液，使用siRNA缀合物7配制2B溶液，每份2B溶液依次含有上述siRNA缀合物7工作液3μl和细胞维持培养基97μl。
配制2C溶液，使用siRNA参比缀合物6配制2C溶液，每份2C溶液依次含有上述siRNA参比缀合物6工作液3μl和细胞维持培养基97μl。
配制2D溶液，使用siRNA参比缀合物7配制2D溶液，每份2D溶液依次含有上述siRNA参比缀合物7工作液3μl和细胞维持培养基97μl。
配制2E溶液，每份2E溶液含有2μl LipofectamineTM 2000和细胞维持培养基98μl。
分别将一份2E溶液与得到的siRNA缀合物6的2A溶液混合，分别室温下孵育20min，得到siRNA缀合物6的转染复合物2Xa。
将一份2E溶液与得到的siRNA缀合物7的2B溶液混合，分别室温下孵育20min，得到siRNA缀合物7的转染复合物2Xb。
将一份2E溶液与得到的siRNA参比缀合物7的2C溶液混合，分别室温 下孵育20min，得到siRNA参比缀合物7的转染复合物2XC。
将一份2E溶液与得到的siRNA参比缀合物7的2D溶液混合，分别室温下孵育20min，得到siRNA参比缀合物7的转染复合物2Xd。
将一份2E溶液与细胞维持培养基100μl混合，在室温下孵育20min，得到空白转染复合物2Xe。
在2个培养孔中，分别加入siRNA缀合物6的转染复合物2Xa，均匀混合，加入量为200μl/孔，得到含siRNA缀合物6终浓度(以siRNA计)约为50nM的转染复合物的转染混合物，记为测试组1。
在2个培养孔中，分别加入siRNA缀合物7的转染复合物2Xb，均匀混合，加入量为200μl/孔，得到含siRNA缀合物7终浓度(以siRNA计)约为50nM的转染复合物的转染混合物，记为测试组2。
在2个培养孔中，分别加入siRNA参比缀合物6的转染复合物2Xc，均匀混合，加入量为200μl/孔，得到含参比siRNA缀合物6终浓度(以siRNA计)约为50nM的转染混合物，记为测试组3。
在2个培养孔中，分别加入siRNA参比缀合物7的转染复合物2Xd，均匀混合，加入量为200μl/孔，得到含参比siRNA缀合物7终浓度(以siRNA计)约为50nM的转染混合物，记为测试组4。
在另外2个培养孔中，分别加入空白转染复合物2Xe，加入量为200μl/孔，得到不含siRNA的转染混合物，记为空白对照组。
将载有含siRNA缀合物6、缀合物7、参比缀合物6和参比缀合物7转染混合物的12孔板置于含5％CO2/95％空气的培养箱在37℃下继续培养24h。
随后，使用UNIQ-10柱式总RNA抽提试剂盒(购自生工公司，货号B511361-0100)根据说明书记载的方法提取各孔细胞中的总RNA，分别得到含总RNA的溶液。
对于每孔细胞，分别取含1μg总RNA的溶液，使用反转录试剂盒GoldenstarTM RT6 cDNA Synthesis Kit(购自北京擎科新业生物技术有限公司，货号TSK301M)提供的试剂，其中选取GoldenstarTM Oligo(dT)17作为引物，按试剂盒说明书中反转录操作步骤配制反转录反应体系20μl，对各孔细胞的总 RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于50℃孵育50min，然后85℃孵育5min，最后4℃孵育30s，反应结束后，向反转录反应体系中加入DEPC水80μl，得到含cDNA的溶液100μL。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μl做模板，使用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HSD和内参基因GAPDH的PCR引物序列如表4所示，每条引物的终浓度为0.25μM。将包含目标基因HSD的各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为在95℃预变性10min，然后在95℃变性30s，在55℃退火30s，在72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因HSD的产物W；对于包含内参基因GAPDH的qPCR体系，使用与上述相同的方法进行qPCR扩增，区别仅在于，扩增程序中退火的温度为60℃，得到含有扩增了内参基因GAPDH的产物W’。产物W和W’随即依次经过95℃15s，60℃1min，95℃15s的孵育，通过实时荧光定量PCR仪分别测定获得产物W和W’中目标基因HSD和内参基因GAPDH的溶解曲线，得到目标基因HSD和内参基因GAPDH的Ct值。
表4：引物信息
对于上述各测试组和对照组，分别进行2次上述定量PCR检测。
采用比较Ct(ΔΔCt)法，对各测试组中目标基因HSD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)是空白对照组两次检测测得的ΔCt(对照组)的算术平均值。从而，各测试组和空白对照组的每一次检测结果均对应一个ΔΔCt值。
以对照组为基准，对测试组HSD mRNA的表达水平进行归一化，定义对照组HSD mRNA表达水平为100％。
测试组HSD mRNA相对表达水平＝2-ΔΔCt(测试组)×100％
测试组HSD mRNA抑制率＝(1-测试组HSD mRNA相对表达水平)×
100％
表5为转染了本公开的siRNA缀合物6和缀合物7后，猴原代肝细胞中HSD mRNA的抑制率。
表5猴原代肝细胞中HSD mRNA的抑制率
由表5的结果可见，在猴原代肝细胞中，本公开提供的siRNA缀合物6和缀合物7在50nM浓度下，与其序列相同、但不包含稳定化修饰核苷酸的参比缀合物相比，显示出显著更高的HSD mRNA抑制活性。
实施例3不同浓度siRNA缀合物在猴肝原代细胞上对HSD mRNA表达量的抑制效率
采用和实验例2中相同的方法制备猴原代肝细胞。
用DEPC水将siRNA缀合物2、缀合物3、缀合物5、和参比缀合物NC分别配制成20μM和2μM(以siRNA计)的siRNA缀合物工作液。
配制3A溶液，每份3A溶液依次含有上述siRNA缀合物2工作液3μl和细胞维持培养基97μl，分别得到3Aa、3Ab的siRNA工作液。
配制3B溶液，每份3B溶液依次含有上述siRNA缀合物3工作液3μl和细胞维持培养基97μl，分别得到3Ba、3Bb的siRNA工作液。
配制3C溶液，每份3C溶液依次含有上述siRNA缀合物5工作液3μl和细胞维持培养基97μl，分别得到3Ca、3Cb的siRNA工作液。
配制3D溶液,每份3D溶液依次含有上述参比缀合物NC工作液3μl和细胞维持培养基97μl，分别得到3Da、3Db的siRNA工作液。
配制3E溶液，每份3E1溶液含有2μl LipofectamineTM 2000和细胞维持培养基98μl。
配制3Xa溶液，分别将1份3E溶液与1份3Aa溶液混合、1份2Ab溶液混合，室温下孵育20min，分别得到转染复合物3Xa1或3Xa2。
配制3Xb溶液，分别将1份3E溶液与1份3Ba溶液混合、1份3Bb溶液混合，室温下孵育20min，分别得到转染复合物3Xb1或3Xb2。
配制3Xc溶液，分别将1份3E溶液与1份3Ca溶液混合、1份3Cb溶液混合，室温下孵育20min，分别得到转染复合物3Xc1或3Xc2。
配制3Xd溶液，分别将1份3E溶液与1份3Da溶液混合、1份3Db溶液混合，室温下孵育20min，分别得到转染复合物3Xd1或3Xd2。
将一份3E溶液与细胞维持培养基100μl混合，在室温下孵育20min，得到空白转染复合物3Xe1。
在2个培养孔，分别加入siRNA缀合物2的转染复合物3Xa1或3Xa2，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为50nM或5nM的转染混合物，记为测试组1。
在2个培养孔中，分别加入siRNA缀合物3的转染复合物3Xb1或3Xb2，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为50nM或5nM的转染混合物，记为测试组2。
在2个培养孔中，分别加入siRNA缀合物5的转染复合物3Xc1或3Xc2，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为50nM或5nM的转染混合物，记为测试组3。
在2个培养孔中，分别加入空白转染复合物3Xe1，加入量为200μl/孔，得到不含siRNA的转染混合物，记为空白对照组。
在2个培养孔中，分别加入siRNA参比缀合物NC的转染复合物3Xf1或3Xf2，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为50nM的转染混合物，记为阴性对照组
将载有含siRNA缀合物2、缀合物3、缀合物5和参比缀合物NC的 转染混合物12孔板置于含5％CO2/95％空气的培养箱在37℃下继续培养24h。
采用和实验例2中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因HSD进行相对定量计算。结果如图1所示。图1是显示了转染50nM和5nM浓度的本公开的siRNA后，体外猴原代肝细胞中的HSD mRNA相对表达水平的柱状图。图1中，blank表示空白对照组，NC表示阴性对照组。由图1的结果可见，在猴原代肝细胞中，本公开提供的siRNA缀合物2、缀合物3和缀合物5在50nM和5nM的siRNA浓度下，显示出高的HSD mRNA抑制活性。
实验例4siRNA缀合物在体外psi-CHECK系统中对脱靶序列的抑制活性
按照实验例1的方法，测试了缀合物3和参比缀合物3在体外psi-CHECK系统中的脱靶序列抑制活性。区别仅在于，对于缀合物3使用如下所示的目标序列4：
目标序列4：
5’-TCGAGTATGATCGTTGCTGAGAGATTGC-3’
(SEQ ID NO:222)
其中，目标序列4中包含与待测缀合物中siRNA反义链部分互补的序列、因此待测缀合物对目标序列4的抑制效果可反应脱靶效应的程度。即，抑制效果越高，表示待测缀合物越可能发生脱靶。
按照实验例1的方法，由该剂量-效应曲线和对应的函数，确定当Y＝50％时对应的X50值，计算获得各siRNA的IC50值＝10^X50(nM)。其结果，在所测定的浓度范围内，缀合物3的脱靶目标序列抑制率始终未低于50％，即，完全未发生脱靶。
表6 siRNA缀合物在psi-CHECK系统中的脱靶效应IC50
由实验例3和实验例4的结果可知，本公开的siRNA缀合物3在大 大提高HSD mRNA抑制活性的同时，表现出比参比缀合物更低的脱靶效应的效果，具有优异的成药能力。
实验例5 siRNA缀合物在小鼠原代肝细胞中的HSD mRNA抑制活性
从C57BL/6j小鼠新鲜肝组织中提取获得小鼠肝原代细胞，在Opti-MEM(1X)培养基(GIBCO公司，货号31985-070)中调整小鼠肝原代细胞密度至1×105细胞/mL，得到小鼠肝原代细胞悬液。随后在12孔板的不同培养孔中分别加入体积为1mL/孔的小鼠肝原代细胞悬液。
用DEPC水将siRNA缀合物6、缀合物7和参比缀合物NC分别配制成20μM(以siRNA计)的siRNA缀合物工作液。
配制5A溶液，每份5A溶液依次含有上述siRNA缀合物6工作液3μl和细胞维持培养基97μl，分别得到5A1的siRNA工作液。
配制5B溶液，每份5B溶液依次含有上述siRNA缀合物7工作液3μl和细胞维持培养基97μl，分别得到5B的siRNA工作液。
配制5C溶液，每份5C溶液依次含有上述参比缀合物NC工作液3μl和细胞维持培养基97μl，分别得到5C的siRNA工作液。
配制5D溶液，每份5D溶液含有2μl LipofectamineTM 2000和细胞维持培养基98μl。
配制5Xa溶液，分别将1份5D溶液与1份5Aa溶液混合，室温下孵育20min，分别得到转染复合物5Xa。
配制5Xb溶液，分别将1份5D溶液与1份5Ba溶液混合，室温下孵育20min，分别得到转染复合物5Xb。
配制5Xc溶液，分别将1份5D溶液与1份5Ca溶液混合，室温下孵育20min，分别得到转染复合物5Xc。
在2个培养孔中，分别加入siRNA缀合物6的转染复合物5Xa，均匀混合，加入量为200μl/孔，得到含siRNA缀合物6终浓度(以siRNA计)约为50nM转染复合物，记为测试组1。
在2个培养孔中，分别加入siRNA缀合物7的转染复合物5Xb，均匀混合，加入量为200μl/孔，得到含siRNA缀合物7终浓度(以siRNA 计)分别约为50nM的转染复合物，记为测试组2。
在2个培养孔中，分别加入siRNA参比缀合物NC转染复合物5Xc，均匀混合，加入量为200μl/孔，得到含对照参比缀合物NC终浓度(以siRNA计)约为50nM的转染复合物，记为阴性对照组。在2个培养孔中，分别加入4D溶液，均匀混合，加入量为200μl/孔，得到含有2μl LipofectamineTM 2000和细胞维持培养基98μl的混合物，记为Lipo对照组。在2个培养孔中，分别加入细胞维持培养基，加入量为200μl/孔，记为空白对照组。
将每一含siRNA的转染混合物和空白对照组置于含5％CO2的培养箱中，在37℃下继续培养24h。
采用和实验例2中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因HSD进行相对定量计算，使用的引物信息如表7所示。结果如图2所示。
表7：引物信息
图2为分别表示缀合物6、缀合物7或参比缀合物NC后，C57BL/6j小鼠原代肝细胞中HSD mRNA相对表达水平的柱状图。其中，blank表示空白对照组，lipo表示Lipo对照组，NC表示阴性对照组。由图2的结果可见，本公开的siRNA缀合物在C57BL/6j小鼠原代肝细胞中显示出优异的HSD mRNA抑制活性，在50nM的siRNA浓度下，HSD mRNA抑制率至少83.11％。
实验例6 siRNA缀合物在小鼠体内的活性效果
将缀合物8和参比缀合物7分别用PBS溶解为3 mg/ml的溶液(以siRNA缀合物计)。将C57BL/6小鼠(雌性，16-18g重，6-8周龄，购自 于斯贝福公司)随机分组，每组6只小鼠，分别编号。以颈背部皮下注射的方式，向每只小鼠分别给予上述siRNA缀合物溶液，给药前称重并记录体重，按体重给药，给药体积均为5mL/kg，作为测试组；另外向一组小鼠中的每只分别给予PBS，给药体积均为5mL/kg，作为空白对照组。
以给药时间点作为第1天计算，在第8天，取测试组和空白对照组每只小鼠的肝组织，用RNAlater保存。
采用和实验例2中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因HSD进行相对定量计算，使用的引物信息如上表7所示。结果如图3所示。
试验数据均以平均数±标准差(Mean±SD)表示，数据分析采用Graphpad prism统计分析软件。数据符合正态分布及方差齐，采用单因素方差分析(one-way由ANOVA)法进行检验；不符合正态分布或方差不齐，采用非参数检验的Kruskal-Wallis H方法进行分析，P≤0.05认为有显著的统计学差异。
图3是给予3mg/kg的缀合物8、参比缀合物7或PBS后，第8天缀合物对小鼠肝细胞中HSD17B13 mRNA的抑制作用。图3可见，与空白对照组相比，给予不包含稳定化修饰核苷酸的参比缀合物7后，小鼠肝细胞中HSD17B13 mRNA的抑制率为57.2％；而给予本公开的siRNA缀合物后，小鼠肝细胞中HSD17B13 mRNA的抑制率为69.34％，显示出比空白对照组和参比缀合物7更高的抑制活性。
实验例7缀合物1在猴原代肝细胞中对HSD mRNA表达量的抑制效率
采用和实验例2中相同的方法得到每孔添加1mL的细胞维持培养基的猴原代肝细胞悬液。
用DEPC水将siRNA缀合物1配制成20μM(以siRNA计)的siRNA缀合物工作液。
配制7A溶液，使用siRNA缀合物1配制7A溶液，每份7A溶液依次含有上述siRNA缀合物1工作液3μl和细胞维持培养基97μl。
配制7B溶液，每份7B溶液含有2μl LipofectamineTM 2000和细胞维持培养基98μl。
分别将一份7B溶液与得到的siRNA缀合物1的7A溶液混合，分别室温下孵育20min，得到siRNA缀合物1的转染复合物7Xa。
将一份7B溶液与细胞维持培养基100μl混合，在室温下孵育20min，得到空白转染复合物7Xb。
在2个培养孔中，分别加入siRNA缀合物1的转染复合物7Xa，均匀混合，加入量为200μl/孔，得到含siRNA缀合物1终浓度(以siRNA计)约为50nM的转染复合物的转染混合物，记为测试组1。
在另外2个培养孔中，分别加入空白转染复合物7Xb，加入量为200μl/孔，得到不含siRNA的转染混合物，记为空白对照组。
将载有含siRNA缀合物1的转染混合物12孔板置于含5％CO2/95％空气的培养箱在37℃下继续培养24h。
采用和实验例2中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因HSD进行相对定量计算，使用的引物信息如上表4所示。
结果显示，本公开提供的包含稳定化修饰核苷酸siRNA缀合物1，在50nM浓度下猴原代肝细胞中HSD mRNA的抑制率达到92.47％，显示出优异的抑制活性。
实验例8 siRNA缀合物3在大鼠体内的抑制活性效果
将缀合物3用PBS溶解为0.6mg/ml的溶液(以siRNA缀合物计)。将将SD大鼠(雄性，200-240g重，6-8周龄，购自于斯贝福公司)，随机分为3组，每组6只，分别编号。以颈背部皮下注射的方式，向每只小鼠分别给予上述siRNA缀合物3溶液，给药前称重并记录体重，按体重给药，给药体积均为5mL/kg，作为测试组；另外向一组小鼠中的每只分别给予PBS，给药体积均为5mL/kg，作为空白对照组。
以给药时间点作为第1天计算，在第8天，取测试组和空白对照组每只大鼠的肝组织，用RNAlater保存。
采用和实验例2中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因HSD进行相对定量计算，其中大鼠内参基因为ACTIN，使用的引物信息如下表8所示。
表8：引物信息
结果显示，给予3mg/kg的缀合物3后，第8天缀合物对大鼠肝细胞中HSD17B13 mRNA的抑制率为74.24％。显示出良好的抑制活性和长效性。
实验例9 siRNA缀合物在猴原代肝细胞中抑制率检测
采用和实验例2中相同的方法得到每孔添加1mL的细胞维持培养基得到猴原代肝细胞悬液。
用DEPC水将siRNA缀合物3、缀合物5、缀合物7、缀合物8、缀合物9、缀合物10和参比缀合物NC分别配制成20μM和2μM(以siRNA计)的siRNA缀合物工作液。
配制9A溶液，每份9A溶液依次含有上述siRNA缀合物3工作液3μl和细胞维持培养基97μl，分别得到9A1、9A2的siRNA工作液。
配制9B溶液，每份9B溶液依次含有上述siRNA缀合物5工作液3μl和细胞维持培养基97μl，分别得到9B1、9B2的siRNA工作液。
配制9C溶液，每份9C溶液依次含有上述siRNA缀合物7工作液3μl和细胞维持培养基97μl，分别得到9C1、9C2的siRNA工作液。
配制9D溶液，每份9D溶液依次含有上述siRNA缀合物8工作液3μl和细胞维持培养基97μl，分别得到9D1、9D2的siRNA工作液。
配制9E溶液，每份9E溶液依次含有上述siRNA缀合物9工作液3μl和细胞维持培养基97μl，分别得到9E1、9E2的siRNA工作液。
配制9F溶液，每份9F溶液依次含有上述siRNA缀合物10工作液3μl和细胞维持培养基97μl，分别得到9F1、9F2的siRNA工作液。配制9G溶液，每份9G溶液含有2μl LipofectamineTM 2000和细胞维持培养基98μl。配制9H溶液，每份9H溶液依次含有上述参比缀合物NC工作液3μl和细胞维持培养基97μl，分别得到9H1、9H2的siRNA工作液。
配制9Xa溶液，分别将1份9G溶液与1份9A1溶液混合、1份9A2溶液混合，室温下孵育20min，分别得到转染复合物9Xa1或9Xa2。
配制9Xb溶液，分别将1份9G溶液与1份9B1溶液混合、1份9B2溶液混合，室温下孵育20min，分别得到转染复合物9Xb1或9Xb2。
配制9Xc溶液，分别将1份9G溶液与1份9C1溶液混合、1份9C2溶液混合，室温下孵育20min，分别得到转染复合物9Xc1或9Xc2。
配制9Xd溶液，分别将1份9G溶液与1份9D1溶液混合、1份9D2溶液混合，室温下孵育20min，分别得到转染复合物9Xd1或9Xd2。
配制9Xe溶液，分别将1份9E溶液与1份9E1溶液混合、1份9E2溶液混合，室温下孵育20min，分别得到转染复合物9Xe1或9Xe2。
配制9Xf溶液，分别将1份9F溶液与1份9F1溶液混合、1份9F2
溶液混合，室温下孵育20min，分别得到转染复合物9Xf1或9Xf2。将一份9G溶液与细胞维持培养基100μl混合，在室温下孵育20min，得到空白转染复合物9Xg。
配制9Xh溶液，分别将1份9H溶液与1份9H1溶液混合、1份9H2溶液混合，室温下孵育20min，分别得到转染复合物9Xh1或9Xh2。
在2个培养孔中，分别加入siRNA缀合物3的转染复合物9Xa1或9Xa2，均匀混合，加入量为200μl/孔，得到含siRNA缀合物3终浓度(以siRNA计)约为50nM或5nM的转染混合物，记为测试组1。
在2个培养孔中，分别加入siRNA缀合物5的转染复合物9Xb1或9Xb2，均匀混合，加入量为200μl/孔，得到含siRNA缀合物5终浓度(以siRNA计)约为50nM或5nM的转染混合物，记为测试组2。
在2个培养孔中，分别加入siRNA缀合物7的转染复合物9Xc1或9Xc2，均匀混合，加入量为200μl/孔，得到含参比siRNA缀合物7终浓度(以siRNA计)约为50nM或5nM的转染混合物，记为测试组3。
在2个培养孔中，分别加入siRNA缀合物8的转染复合物9Xd1或9Xd2，均匀混合，加入量为200μl/孔，得到含参比siRNA缀合物8终浓度(以siRNA计)约为50nM或5nM的转染混合物，记为测试组4。
在2个培养孔中，分别加入siRNA缀合物9的转染复合物9Xe1或9Xe2，均匀混合，加入量为200μl/孔，得到含参比siRNA缀合物9终浓度(以siRNA计)约为50nM或5nM的转染混合物，记为测试组5。
在2个培养孔中，分别加入siRNA缀合物10的转染复合物9Xf1或9Xf2，均匀混合，加入量为200μl/孔，得到含参比siRNA缀合物10终浓度(以siRNA计)约为50nM或5nM的转染混合物物，记为测试组6。在另外2个培养孔中，分别加入空白转染复合物2Xg，加入量为200μl/孔，得到不含siRNA的转染混合物，记为空白对照组。
在2个培养孔中，分别加入siRNA参比缀合物NC的转染复合物3Xh1或3Xh2，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为50nM的转染混合物，记为阴性对照组。
将载有含siRNA缀合物3、缀合物5、缀合物7、缀合物8、缀合物9、缀合物10和参比缀合物NC的转染混合物的12孔板置于含5％CO2/95％空气的培养箱在37℃下继续培养24h。
采用和实验例2中相同的方法提取各孔细胞中的总RNA和反转录并对各测试组中目标基因HSD进行相对定量计算，使用的引物信息如上表4所示。结果如图4所示，blank表示空白对照组，NC表示阴性对照组
由图4的结果可见，在猴原代肝细胞中，本公开提供的siRNA缀合物包含1个碱基错配的缀合物与不包含1个碱基错配的缀合物，均显示出优异的HSD mRNA抑制活性。
实验例10 siRNA缀合物在HEK293A细胞SEAP-HSD模型的体外活性
用DEPC水将siRNA缀合物3、缀合物5、缀合物7、缀合物8、缀合物9和缀合物10分别配制成0.12μM和0.012μM(以siRNA计)的siRNA缀合物工作液。
配置10A溶液，每份10A溶液含有2μl LipofectamineTM 2000和细胞维持培养基98μl。
将1份10A溶液100μl分别与上述siRNA缀合物3工作液100μl混合，室温下孵育20min，分别得到转染复合物10X1和10X01。
将1份10A溶液100μl分别与上述siRNA缀合物5工作液100μl混合，室温下孵育20min，分别得到转染复合物10X2和10X02。
将1份10A溶液100μl分别与上述siRNA缀合物7工作液100μl混合，室温下孵育20min，分别得到转染复合物10X3和10X03。
将1份10A溶液100μl分别与上述siRNA缀合物8工作液100μl混合，室温下孵育20min，分别得到转染复合物10X4和10X04。
将1份10A溶液100μl分别与上述siRNA缀合物9工作液100μl混合，室温下孵育20min，分别得到转染复合物10X5和10X05。
将1份10A溶液100μl分别与上述siRNA缀合物10工作液100μl混合，室温下孵育20min，分别得到转染复合物10X6和10X06。
将1份10A溶液与细胞维持培养基100μl混合，在室温下孵育20min，得到对照组转染复合物10X。
取对数生长期的HEK293A细胞，按照1×105细胞/孔进行铺板，每孔1mL。实验前吸尽培养孔中完全培养基，每孔加入1mL opti-MEM培养基(GIBCO公司)。在2个培养孔，分别加入100ng/μl的SEAP-HSD质粒溶液，每孔1μl；以及siRNA缀合物3的转染复合物10X1或10X01，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为10nM或1nM的共转染混合物，记为测试组1。
在2个培养孔，分别加入加入100ng/μl的SEAP-HSD质粒溶液， 每孔1μl；以及siRNA缀合物5的转染复合物10X2或10X02，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为10nM或1nM的共转染混合物，记为测试组2。
在2个培养孔，分别加入100ng/μl的SEAP-HSD质粒溶液，每孔1μl；以及加入siRNA缀合物7的转染复合物10X3或10X03，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为10nM或1nM的共转染混合物，记为测试组3。
在2个培养孔，分别加入加入100ng/μl的SEAP-HSD质粒溶液，每孔1μl；以及siRNA缀合物8的转染复合物10X4或10X04，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为10nM或1nM的共转染混合物，记为测试组4。
在2个培养孔，分别加入加入100ng/μl的SEAP-HSD质粒溶液，每孔1μl；以及siRNA缀合物9的转染复合物10X5或10X05，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为10nM或1nM的共转染混合物，记为测试组5。
在2个培养孔，分别加入加入100ng/μl的SEAP-HSD质粒溶液，每孔1μl；以及siRNA缀合物10的转染复合物10X6或10X06，均匀混合，加入量为200μl/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为10nM或1nM的共转染混合物，记为测试组6。
在2个培养孔中，分别加入100ng/μl的SEAP-HSD质粒溶液，每孔1μl；以及对照组转染复合物10X，加入量为200μl/孔，得到不含siRNA的转染混合物，记为对照组。
将载有含siRNA缀合物3、缀合物5、缀合物7、缀合物8、缀合物9和缀合物10的转染混合物的12孔板置于含5％CO2/95％空气的培养箱在37℃下继续培养24h。将所述检测质粒与待测缀合物共转染至HEK293A细胞中，通过SEAP酶活的水平，来反映siRNA的目标序列抑制活性。上述SEAP-HSD质粒含有插入SEAP(分泌型碱性磷酸酶，购自北京原平皓生物技术有限公司)报告基因的3'UTR内的HSD cDNA序列(Genbank注册号NM_001136230.3)。
根据说明书，SEAP Reporter GENE ASSAY KIT(Luminescence)abcam-ab 133077Protocol对上述细胞进行测量。每个测试组SEAP水平用对照组样本进行标准化。具体来说，首先，将每个测试组在各时间点的SEAP水平除以各时间点对照组SEAP水平，从而确定给予缀合物后的相对表达水平。根据各组细胞样本在各个时间点的SEAP相对表达水平的算术平均值S，siRNA对SEAP-HSD酶活的抑制率＝(1-S)×100％。
按照上述方法计算siRNA对HSD mRNA的抑制率，如下表9所示。
表9猴原代肝细胞中HSD mRNA的抑制率
图5显示了在10nM和1nM浓度下，HEK293A细胞SEAP浓度的柱状图。
结果表明，与空白对照组相比，在24h和72h，各个测试组的SEAP均明显降低。其中，缀合物9在72h表现出特别高的HSD mRNA的抑制率，10nM浓度下抑制率达到91.6％，1nM浓度下抑制率达到89.9％。72小时对HSD17B13基因表达的mRNA的抑制活性最高可达到92.4％，各个组别72小时的抑制率整体高于24小时的抑制率，显示出对HSD17B13mRNA长效的抑制活性。
实验例11 siRNA缀合物在大鼠的体内抑制活性
将SD大鼠(雄性，200-240g重，6-8周龄，购自于斯贝福公司)，随机分为3组，每组5只。
用PBS将上述制备的参比缀合物3和缀合物9用PBS分别配制为0.6mg/mL的溶液(以siRNA计)。以颈背部皮下注射的方式，向每组小鼠分别给予上述siRNA缀合物溶液，给药前称重并记录体重，按体重给药，单次给药容积为5μL/g小鼠体重，计算可知单次给药剂量为3mg/kg。 另外向一组每只小鼠分别给予PBS，单次给药体积为5μL/g，作为空白对照组。
以给药时间点作为第1天计算，在第8天和第15天，取测试组和空白对照组每只大鼠的肝组织，用RNAlater保存。
采用和实验例2中相同的方法提取各孔细胞中的总RNA和反转录并以空白对照组的结果归一化，对各测试组中目标基因HSD进行相对定量计算，使用的引物信息如上表8所示。
试验数据均以平均数±标准差(Mean±SD)表示，数据分析采用Graphpad prism统计分析软件。数据符合正态分布及方差齐，采用单因素方差分析(one-way ANOVA)法进行检验；不符合正态分布或方差不齐，采用非参数检验的Kruskal Wallis H方法进行分析，P≤0.05认为有显著的统计学差异。结果如图6所示。
图6是显示了在给予本公开的3 mg/kg的siRNA缀合物后，第8天和第15天缀合物对小鼠肝细胞中HSD17B13 mRNA的抑制率。其中，“对比缀合物3”表示参比缀合物3的结果，“PBS”表示空白对照组的结果。图6的结果显示，给予本公开的包含稳定化修饰核苷酸的siRNA缀合物后，小鼠肝细胞中HSD17B13 mRNA的抑制率明显高于参比缀合物，显示出更高的抑制活性。其中，在第8天时，HSD17B13 mRNA的抑制率为81.2％，与不包含稳定化修饰核苷酸的参比缀合物相比，表现出更加优异的HSD17B13 mRNA抑制活性；在第15天时更是显著高于对比缀合物，表明本公开的siRNA缀合物还具有优异的体内HSD17B13mRNA长效抑制活性。
实验例12本实验说明本发明的siRNA缀合物的动物水平毒性。
将SD大鼠(购自斯贝福(北京)生物技术有限公司)分为5组，每组10只，雌雄各半。
向每组大鼠分别皮下单次给予30mg/kg或100mg/kg(以siRNA计)的本公开的缀合物3、缀合物9和PBS为对照组，给药前称重并记录体重，按体重给药，给药当天记为D1，分别于D15，D29给药，共给药3次，D30天进行组织病理学检查。
观察期间大鼠表现无死亡或行为异常，各剂量组动物和对照组动物，体重增长均无显著性差异。
组织病理学检查：对大鼠进行解剖，剖取肝组织，经取材、脱水、包埋、制片和染色后，制成病理切片，在光学显微镜下(显微镜型号：NIKON Eclipse ci，成像系统：NIKON digital sight DS-FI2，MADE IN JAPAN)观察。其结果，给予缀合物3和缀合物9的大鼠的肝脏病理切片中，组织肝索结构清晰，肝细胞排列紧密，界限清晰，胞质丰富，着色均匀，胞核圆形，大小正常，静脉内皮完整正常，组织未见明显异常。结果显示，各剂量组雌性和雄性动物各脏器重量均无显著性改变。由此可见，本公开的缀合物毒性小，体内耐受性良好，因此相对于显示出药效的剂量(3 mg/kg动物体重)，具有至少30倍以上的安全窗口，适合作为体内抑制HSD17B13mRNA的药物使用。
上述结果表明，与参比缀合物相比，本公开的siRNA缀合物具有更加有效抑制HSD17B13 mRNA的作用，因此在制备用于HSD疾病或症状的治疗和/或预防的药物方面显示出显著更高的治疗效果，具有优异的开发前景。
以上详细描述了本公开的一些实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述一些实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (43)

  1. 一种siRNA，所述siRNA包含正义链和反义链，所述正义链包含核苷酸序列I，所述反义链包含核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II均由19个核苷酸组成，所述核苷酸序列I和所述核苷酸序列II中的每一个核苷酸均为修饰或未修饰的核苷酸，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，所述核苷酸序列II至少部分地与第一段核苷酸序列反向互补，所述第一段核苷酸序列为HSD17B13基因表达的mRNA中的一段长度为19个核苷酸的核苷酸序列，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-6个核苷酸中的至少1个为稳定化修饰核苷酸，所述稳定化修饰核苷酸指核苷酸的核糖2'位羟基被稳定化修饰基团取代的核苷酸，与相应位置的核苷酸为未修饰的核苷酸的siRNA相比，包含所述稳定化修饰核苷酸的siRNA的热稳定性增加，并且所述稳定化修饰基团的空间位阻大于2'-O-甲基。
  2. 如权利要求1所述的siRNA，其中，按照5'末端到3'末端的方向，所述核苷酸序列II中的第3个或第5个核苷酸为所述稳定化修饰核苷酸。
  3. 如权利要求1或2所述的siRNA，其中，按照5'末端到3'末端的方向，所述核苷酸序列II中第3-9个核苷酸中不超过2个核苷酸为所述稳定化修饰核苷酸。
  4. 如权利要求1-3中任意一项所述的siRNA，其中，所述siRNA的热稳定性增加是指所述siRNA的Tm升高，Tm为siRNA的双链热解离温度。
  5. 如权利要求4所述的siRNA，其中，所述siRNA的热稳定性增加是指所述siRNA的Tm升高至少0.05℃。
  6. 如权利要求4所述的siRNA，其中，所述siRNA的热稳定性增加是指所述siRNA的Tm升高0.1-6℃。
  7. 如权利要求4所述的siRNA，其中，所述siRNA的热稳定性增加是指所述siRNA的Tm升高0.5-4℃。
  8. 如权利要求1-7中任意一项所述的siRNA，其中，每个所述稳定化修饰基团独立地具有-X-R所示的结构，其中，X为O、NR'、S或SiR'2；R是C2-C6烷基、取代的C2-C6烷基、C6-C8芳基、取代的C6-C8芳基中的一种，每个R'独立地是H、C1-C6烷基、取代的C1-C6烷基、C6-C8芳基、取代的C6-C8芳基中的一种，所述取代的C2-C6烷基、取代的C6-C8芳基或取代的C1-C6烷基是指C2-C6烷基、C6-C8芳基或C1-C6烷基中的一个或多个氢原子被取代基取代而形成的基团，所述取代基选自以下取代基中的一种或多种：C1-C3烷基、C6-C8芳基、C1-C3烷氧基、卤素、氧亚基和硫亚基。
  9. 如权利要求8所述的siRNA，其中，每个所述稳定化修饰基团独立地选自2'-O-甲氧基乙基、2'-O-烯丙基、2'-烯丙基、2'-O-2-N-甲基氨基-2-氧亚基乙基、2'-O-2-N,N-二甲基氨基乙基、2'-O-3-氨基丙基和2'-O-2,4-二硝基苯基中的一种。
  10. 如权利要求9所述的siRNA，其中，每个所述稳定化修饰基团为2'-O-甲氧基乙基。
  11. 如权利要求1-10中任意一项所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GAAUAAUGCUGGGACAGUZa1-3'(SEQ ID NO:1)；
    5'-Za2ACUGUCCCAGCAUUAUUC-3'(SEQ ID NO:2),
    其中，所述Za1为A,Za2为U，所述核苷酸序列I中包含位置对应于Za1的核苷酸Za3，所述核苷酸序列II中包含位置对应于Za2的核苷酸Za4，所述Za4是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:27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:28所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GCACCAAGGAUGAAGAGAZb1-3'(SEQ ID NO:27)；
    5'-Zb2UCUCUUCAUCCUUGGUGC-3'(SEQ ID NO:28),
    其中Zb1为U,Zb2为A，所述核苷酸序列I中包含位置对应于Zb1的核苷酸Zb3，所述核苷酸序列II中包含位置对应于Zb2的核苷酸Zb4，所述Zb4是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:53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:54所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-CACCAAGGAUGAAGAGAUZc1-3'(SEQ ID NO:53)；
    5'-Zc2AUCUCUUCAUCCUUGGUG-3'(SEQ ID NO:54),
    其中Zc1为U,Zc2为A，所述核苷酸序列I中包含位置对应于Zc1的核苷酸Zc3，所述核苷酸序列II中包含位置对应于Zc2的核苷酸Zc4，所述Zc4是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:79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:80所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-UCUGAUAGAUGGAAUACUZd1-3'(SEQ ID NO:79)；
    5'-Zd2AGUAUUCCAUCUAUCAGA-3'(SEQ ID NO:80),
    其中Zd1为U,Zd2为A，所述核苷酸序列I中包含位置对应于Zd1的核苷酸Zd3，所述核苷酸序列II中包含位置对应于Zd2的核苷酸Zd4，所述Zd4是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:105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:106所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-CUGAUAGAUGGAAUACUUZe1-3'(SEQ ID NO:105)；
    5'-Ze2AAGUAUUCCAUCUAUCAG-3'(SEQ ID NO:106),
    其中Ze1为A,Ze2为U，所述核苷酸序列I中包含位置对应于Ze1的核苷酸Ze3，所述核苷酸序列II中包含位置对应于Ze2的核苷酸Ze4，所 述Ze4是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:131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:132所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GAUGGAAUACUUACCAAUZf1-3'(SEQ ID NO:131)；
    5'-Zf2AUUGGUAAGUAUUCCAUC-3'(SEQ ID NO:132),
    其中Zf1为A,Zf2为U，所述核苷酸序列I中包含位置对应于Zf1的核苷酸Zf3，所述核苷酸序列II中包含位置对应于Zf2的核苷酸Zf4，所述Zf4是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:157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:158所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GAAUACUUACCAAUAAGAZg1-3'(SEQ ID NO:157)；
    5'-Zg2UCUUAUUGGUAAGUAUUC-3'(SEQ ID NO:158),
    其中Zg1为A,Zg2为U，所述核苷酸序列I中包含位置对应于Zg1的核苷酸Zg3，所述核苷酸序列II中包含位置对应于Zg2的核苷酸Zg4，所述Zg4是所述反义链5'末端的第一个核苷酸。
  12. 如权利要求11所述的siRNA，其中，所述核苷序列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:27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:28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:53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:54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:79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:80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:105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:106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:131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:132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:157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:158所示的核苷酸序列之间不多于1个核苷酸差异。
  13. 如权利要求12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差异包括Za4位置处的差异，且Za4选自A、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:28所示的核苷酸序列之间的差异包括Zb4位置处的差异，且Zb4选自U、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:54所示的核苷酸序列之间的差异包括Zc4位置处的差异，且Zc4选自U、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:80所示的核苷酸序列之间的差异包括Zd4位置处的差异，且Zd4选自U、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:106所示的核苷酸序列之间的差异包括Ze4位置处的差异，且Ze4选自A、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:132所示的核苷酸序列之间的差异包括Zf4位置处的差异，且Zf4选自A、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:158所示的核苷酸序列之间的差异包括Zg4位置处的差异，且Zg4选自A、G或C。
  14. 如权利要求1-13中任意一项所述的siRNA，其中，所述Za3是与Za4互补的核苷酸；或者所述Zb3是与Zb4互补的核苷酸；或者所述Zc3是与Zc4互补的核苷酸；或者所述Zd3是与Zd4互补的核苷酸；或者所述Ze3是与Ze4互补的核苷酸；或者所述Zf3是与Zf4互补的核苷酸；或者所述Zg3是与Zg4互补的核苷酸。
  15. 如权利要求1-14中任意一项所述的siRNA，其中，所述核苷酸序列II与所述第一段核苷酸序列基本上反向互补、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个碱基的错配；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个碱基的错配；所述完全反向互补是指两个核苷酸序列之间没有错配。
  16. 如权利要求15所述的siRNA，其中，按照5'末端到3'末端的方向，所述核苷酸序列II的第3-19位的核苷酸与所述第一段核苷酸序列第1-17位的核苷酸完全反向互补。
  17. 如权利要求16所述的siRNA，其中，所述核苷酸序列II与所述核苷酸序列I完全反向互补；或者按照5'末端到3'末端的方向，所述核苷酸序列II中的第2个核苷酸与按照3'末端到5'末端的方向，所述核苷酸序列I中的第2个核苷酸之间存在碱基错配。
  18. 如权利要求1-17中任意一项所述的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。
  19. 如权利要求1-18中任意一项所述的siRNA，其中，所述核苷酸序列II中的全部核苷酸均为修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列II的第2、6、14、16个核苷酸，如果不是所述稳定化修饰核苷酸的话，为氟代修饰的核苷酸，所述核苷酸序列II中的其它核苷酸各自独立地为非氟代修饰的核苷酸中的一种。
  20. 如权利要求1-19中任意一项所述的siRNA，其中，按照5'末端到3'末端的方向，所述核苷酸序列I的第7-9个核苷酸为氟代修饰的核苷酸。
  21. 如权利要求20所述的siRNA，其中，所述核苷酸序列I中的全部 核苷酸均为修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列I的第7-9个核苷酸为氟代修饰的核苷酸，所述核苷酸序列I中的其它核苷酸各自独立地为非氟代修饰的核苷酸中的一种。
  22. 如权利要求1-21中任意一项所述的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，所述核苷酸序列III和所述核苷酸序列IV的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是所述稳定化修饰核苷酸，所述核苷酸序列III的长度为1个、2个、3个或4个核苷酸，所述核苷酸序列IV和所述核苷酸序列III长度相等，并且所述核苷酸序列IV和所述核苷酸序列III实质上反向互补或完全反向互补，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，并且所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或完全反向互补，该第二段核苷酸序列是指和HSD17B13基因表达的mRNA中与第一段核苷酸序列的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。
  23. 如权利要求22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个核苷酸的差异，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，核苷酸序列IV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，所述核苷酸序列III的碱基组成为GU，所述核苷酸序列IV的碱基组成为AC；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为GGU，所述核苷酸序列IV的碱基组成为ACC；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸序列III的碱基组成为UGGU，所述核苷酸序列IV的碱基组成为ACCA；
    或者，所述核苷酸序列I与SEQ ID NO:27所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，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，核苷酸序列IV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，所述核苷酸序列III的碱基组成为CA，所述核苷酸序列IV的碱基组成为 UG；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为UCA，所述核苷酸序列IV的碱基组成为UGA；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸III的碱基组成为CUCA，所述核苷酸序列IV的碱基组成为UGAG；
    或者，所述核苷酸序列I与SEQ ID NO:53所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，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，核苷酸序列IV的碱基为C；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为AG，所述核苷酸序列IV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，所述核苷酸序列III的碱基组成为CAG，所述核苷酸序列IV的碱基组成为CUG；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸III的碱基组成为UCAG，所述核苷酸序列IV的碱基组成为CUGA；
    或者，所述核苷酸序列I与SEQ ID NO:79所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，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，核苷酸序列IV的碱基为C；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为AG，所述核苷酸序列IV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，所述核苷酸序列III的碱基组成为AAG，所述核苷酸序列IV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，所述核苷酸III的碱基组成为GAAG，所述核苷酸序列IV的碱基组成为CUUC；
    或者，所述核苷酸序列I与SEQ ID NO:105所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，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，核苷酸序列IV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，所述核苷酸序列III的碱基组成为GU，所述核苷酸序列IV的碱基组成为AC；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为AGU，所述核苷酸序列IV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，所述核苷酸III的碱基组成为AAGU，所述核苷酸序列IV的碱基组成为ACUU；
    或者，所述核苷酸序列I与SEQ ID NO:131所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，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，核苷酸序列IV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，所述核苷酸序列III的碱基组成为UA，所述核苷酸序列IV的碱基组成为UA；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为AUA，所述核苷酸序列IV的碱基组成为UAU；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸III的碱基组成为GAUA，所述核苷酸序列IV的碱基组成为UAUC；
    或者，所述核苷酸序列I与SEQ ID NO:157所示的核苷酸序列长度相等，且不多于3个核苷酸的差异，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，核苷酸序列IV的碱基为C；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为UG，所述核苷酸序列IV的碱基组成为CA；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为AUG，所述核苷酸序列IV的碱基组成为CAU；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸III的碱基组成为GAUG，所述核苷酸序列IV的碱基组成为CAUC。
  24. 如权利要求1-23中任意一项所述的siRNA，其中所述siRNA还含有寡核苷酸序列V，所述寡核苷酸序列V的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是所述稳定化修饰核苷酸，所述核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3'末端，从而构成所述反义链的3'突出端。
  25. 如权利要求24所述的siRNA，其中所述核苷酸序列V的长度为2个核苷酸，并且按照5'末端到3'末端的方向，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、连续的2个尿嘧啶核糖核苷酸、或者与第三段核苷酸序列完全反向互补的序列，所述第三段核苷酸序列是指HSD17B13基因表达的mRNA中与第一段核苷酸序列的5'末端或第二段核苷酸序列的5'末端相邻、并且长度与所述核苷酸序列V相等的核苷 酸序列。
  26. 如权利要求25所述的siRNA，其中，所述第一段核苷酸序列具有SEQ ID NO:1所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是GU；或者，所述第一段核苷酸序列具有SEQ ID NO:27所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是CA；或者，所述第一段核苷酸序列具有SEQ ID NO:53所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是AG；或者，所述第一段核苷酸序列具有SEQ ID NO:79所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是AG；或者，所述第一段核苷酸序列具有SEQ ID NO:105所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是GU；或者，所述第一段核苷酸序列具有SEQ ID NO:131所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是UA；或者，所述第一段核苷酸序列具有SEQ ID NO:157所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是UG。
  27. 如权利要求1-26中任意一项所述的siRNA，其中，所述siRNA的正义链含有如SEQ ID NO:3所示的核苷酸序列，所述反义链含有如SEQ ID NO:4所示的核苷酸序列：
    5'-GAACAGAGAUACUACGGUZa3-3'(SEQ ID NO:3)；
    5'-Za4ACUGUCCCAGCAUUAUUCAC-3'(SEQ ID NO:4),
    其中，所述Za4是反义链5'末端的第一个核苷酸，Za3选自A、U、G或C，并且Za3是与Za4互补的核苷酸；
    或者，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'-GUGAAUAAUGCUGGGACAGUZa3-3'(SEQ ID NO:5)；
    5'-Za4ACUGUCCCAGCAUUAUUCACCA-3'(SEQ ID NO:6),
    其中，所述Za4是反义链5'末端的第一个核苷酸，Za3选自A、U、G或C，并且Za3是与Za4互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:29所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:30所示的核苷酸序列：
    5'-GCACCAAGGAUGAAGAGAZb3-3'(SEQ ID NO:29)；
    5'-Zb4UCUCUUCAUCCUUGGUGCUG-3'(SEQ ID NO:30),
    其中，所述Zb4是反义链5'末端的第一个核苷酸，Zb3选自A、U、G或C，并且Zb4是与Zb3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:31所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:32所示的核苷酸序列：
    5'-CAGCACCAAGGAUGAAGAGAZb3-3'(SEQ ID NO:31)；
    5'-Zb4UCUCUUCAUCCUUGGUGCUGAG-3'(SEQ ID NO:32),
    其中，所述Zb4是反义链5'末端的第一个核苷酸，Zb3选自A、U、G或C，并且Zb4是与Zb3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:55所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:56所示的核苷酸序列：
    5'-CACCAAGGAUGAAGAGAUZc3-3'(SEQ ID NO:55)；
    5'-Zc4AUCUCUUCAUCCUUGGUGCU-3'(SEQ ID NO:56),
    其中，所述Zc4是反义链5'末端的第一个核苷酸，Zc3选自A、U、G或C，并且Zc4是与Zc3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:57所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:58所示的核苷酸序列：
    5'-AGCACCAAGGAUGAAGAGAUZc3-3'(SEQ ID NO:57)；
    5'-Zc4AUCUCUUCAUCCUUGGUGCUGA-3'(SEQ ID NO:58),
    其中，所述Zc4是反义链5'末端的第一个核苷酸，Zc3选自A、U、G或C，并且Zc4是与Zc3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:81所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:82所示的核苷酸序列：
    5'-UCUGAUAGAUGGAAUACUZd3-3'(SEQ ID NO:81)；
    5'-Zd4AGUAUUCCAUCUAUCAGACU-3'(SEQ ID NO:82),
    其中，所述Zd4是反义链5'末端的第一个核苷酸，Zd3选自A、U、G或C，并且Zd4是与Zd3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:83所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:84所示的核苷酸序列：
    5'-AGUCUGAUAGAUGGAAUACUZd3-3'(SEQ ID NO:83)；
    5'-Zd4AGUAUUCCAUCUAUCAGACUUC-3'(SEQ ID NO:84),
    其中，所述Zd4是反义链5'末端的第一个核苷酸，Zd3选自A、U、G或C，并且Zd4是与Zd3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:107所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:108所示的核苷酸序列：
    5'-CUGAUAGAUGGAAUACUUZe3-3'(SEQ ID NO:107)；
    5'-Ze4AAGUAUUCCAUCUAUCAGAC-3'(SEQ ID NO:108),
    其中，所述Ze4是反义链5'末端的第一个核苷酸，Ze3选自A、U、G或C，并且Ze4是与Ze3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:109所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:110所示的核苷酸序列：
    5'-GUCUGAUAGAUGGAAUACUUZe3-3'(SEQ ID NO:109)；
    5'-Ze4AAGUAUUCCAUCUAUCAGACUU-3'(SEQ ID NO:110),
    其中，所述Ze4是反义链5'末端的第一个核苷酸，Ze3选自A、U、G或C，并且Ze4是与Ze3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:133所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:134所示的核苷酸序列：
    5'-GAUGGAAUACUUACCAAUZf3-3'(SEQ ID NO:133)；
    5'-Zf4AUUGGUAAGUAUUCCAUCUA-3'(SEQ ID NO:134),
    其中，所述Zf4是反义链5'末端的第一个核苷酸，Zf3选自A、U、G或C，并且Zf4是与Zf3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:135所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:136所示的核苷酸序列：
    5'-UAGAUGGAAUACUUACCAAUZf3-3'(SEQ ID NO:135)；
    5'-Zf4AUUGGUAAGUAUUCCAUCUAUC-3'(SEQ ID NO:136),
    其中，所述Zf4是反义链5'末端的第一个核苷酸，Zf3选自A、U、G或C，并且Zf4是与Zf3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:159所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:160所示的核苷酸序列：
    5'-GAAUACUUACCAAUAAGAZg3-3'(SEQ ID NO:159)；
    5'-Zg4UCUUAUUGGUAAGUAUUCCA-3'(SEQ ID NO:160),
    其中，所述Zg4是反义链5'末端的第一个核苷酸，Zg3选自A、U、G 或C，并且Zg4是与Zg3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:161所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:162所示的核苷酸序列：
    5'-UGGAAUACUUACCAAUAAGAZg3-3'(SEQ ID NO:161)；
    5'-Zg4UCUUAUUGGUAAGUAUUCCAUC-3'(SEQ ID NO:162),
    其中，所述Zg4是反义链5'末端的第一个核苷酸，Zg3选自A、U、G或C，并且Zg4是与Zg3互补的核苷酸。
  28. 如权利要求1-27中任意一项所述的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。
  29. 如权利要求28所述的siRNA，其中，不多于3个非氟代修饰的核苷酸为2'-脱氧核苷酸，其余每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸；或者，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。
  30. 如权利要求29所述的siRNA，其中，所述siRNA为siHSDa1-M1、siHSDa1-M2、siHSDa2-M1、siHSDa2-M2、siHSDb1-M1、siHSDb1-M2、siHSDb2-M1、siHSDb2-M2、HSDc1-M1、siHSDc1-M2、siHSDc2-M1、siHSDc2-M2、HSDd1-M1、siHSDd1-M2、siHSDd2-M1、siHSDd2-M2、siHSDe1-M1、siHSDe1-M2、siHSDe2-M1、siHSDe2-M2、siHSDf1-M1、siHSDf1-M2、siHSDf2-M1、siHSDf2-M2、siHSDg1-M1、siHSDg1-M2、siHSDg2-M1或siHSDg2-M2中的一种。
  31. 如权利要求1-30中任意一项所述的siRNA，其中，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的至少1个磷酸酯基为具有修饰基团的磷酸酯基，所述具有修饰基团的磷酸酯基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'末端端部第1个核苷酸和第2个核苷酸之间；
    所述正义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
    所述正义链的3'末端端部第1个核苷酸和第2个核苷酸之间；
    所述正义链的3'末端端部第2个核苷酸和第3个核苷酸之间；
    所述反义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
    所述反义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
    所述反义链的3'末端端部第1个核苷酸和第2个核苷酸之间；以及
    所述反义链的3'末端端部第2个核苷酸和第3个核苷酸之间。
  32. 如权利要求31所述的siRNA，其中，所述siRNA为siHSDa1-M1S、siHSDa1-M2S、siHSDa2-M1S、siHSDa2-M2S、siHSDb1-M1S、siHSDb1-M2S、siHSDb2-M1S、siHSDb2-M2S、HSDc1-M1S、siHSDc1-M2S、siHSDc2-M1S、siHSDc2-M2S、HSDd1-M1S、siHSDd1-M2S、siHSDd2-M1S、siHSDd2-M2S、siHSDe1-M1S、siHSDe1-M2S、siHSDe2-M1S、siHSDe2-M2S、siHSDf1-M1S、siHSDf1-M2S、siHSDf2-M1S、siHSDf2-M2S、siHSDg1-M1S、siHSDg1-M2S、siHSDg2-M1S或siHSDg2-M2S中的一种。
  33. 如权利要求1-32中任意一项所述的siRNA，其中，所述反义链的5'末端核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。
  34. 如权利要求33所述的siRNA，其中，所述siRNA为siHSDa1-M1P、siHSDa1-M2P、siHSDa2-M1P、siHSDa2-M2P、siHSDb1-M1P、siHSDb1-M2P、siHSDb2-M1P、siHSDb2-M2P、HSDc1-M1P、siHSDc1-M2P、siHSDc2-M1P、siHSDc2-M2P、HSDd1-M1P、siHSDd1-M2P、siHSDd2-M1P、siHSDd2-M2P、siHSDe1-M1P、siHSDe1-M2P、siHSDe2-M1P、siHSDe2-M2P、siHSDf1-M1P、siHSDf1-M2P、siHSDf2-M1P、siHSDf2-M2P、siHSDg1-M1P、siHSDg1-M2P、siHSDg2-M1P或siHSDg2-M2P中的一种。
  35. 一种药物组合物，该药物组合物含有如权利要求1-34中任意一项所述的siRNA以及药学上可接受的载体。
  36. 一种siRNA缀合物，所述siRNA缀合物含有权利要求1-34中任 意一项所述的siRNA以及缀合连接至该siRNA的缀合基团，所述缀合基团包含接头和药学上可接受的靶向基团，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，每个所述靶向基团选自能够和细胞表面受体结合的配体。
  37. 如权利要求1-34中任意一项所述的siRNA，和/或权利要求35所述的药物组合物和/或权利要求36所述的siRNA缀合物在制备用于治疗和/或预防与HSD17B13基因表达的mRNA水平相关的疾病或者症状的药物中的用途。
  38. 如权利要求37所述的用途，其中所述与HSD17B13基因表达的mRNA水平相关的疾病或症状是慢性纤维炎性肝病。
  39. 如权利要求38所述的用途，所述慢性纤维炎性肝病选自由下列所组成的组：肝炎、肝纤维化、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪性肝病(NAFLD)、肝硬化、酒精性脂肪性肝炎(ASH)、酒精性脂肪肝病(ALD)、丙型肝炎(HCV)相关的硬化、药物引起的肝损伤及肝细胞坏死。
  40. 一种治疗和/或预防与HSD17B13基因表达的mRNA水平相关的疾病或症状的方法，所述方法包括向有需要的受试者给予权利要求1-34中任意一项所述的siRNA，和/或权利要求35所述的药物组合物和/或权利要求36所述的siRNA缀合物。
  41. 如权利要求40所述的方法，其中，所述与HSD17B13基因表达的mRNA水平相关的疾病或症状是慢性纤维炎性肝病。
  42. 一种抑制细胞中HSD17B13基因表达水平的方法，所述方法包括将有效剂量的权利要求1-34中任意一项所述的siRNA，和/或权利要求35所述的药物组合物和/或权利要求36所述的siRNA缀合物与所述细胞接触。
  43. 一种试剂盒，所述试剂盒包含权利要求1-34中任意一项所述的siRNA，和/或权利要求35所述的药物组合物和/或权利要求36所述的siRNA缀合物。
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