WO2023116764A1 - 一种核酸、含有该核酸的组合物与缀合物及其用途 - Google Patents

一种核酸、含有该核酸的组合物与缀合物及其用途 Download PDF

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WO2023116764A1
WO2023116764A1 PCT/CN2022/140698 CN2022140698W WO2023116764A1 WO 2023116764 A1 WO2023116764 A1 WO 2023116764A1 CN 2022140698 W CN2022140698 W CN 2022140698W WO 2023116764 A1 WO2023116764 A1 WO 2023116764A1
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nucleotide sequence
nucleotide
seq
sirna
nucleotides
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French (fr)
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梁子才
张鸿雁
高山
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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

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  • the present disclosure relates to a siRNA, a pharmaceutical composition and a conjugate containing the siRNA and uses thereof. Specifically, the present disclosure relates to a siRNA for inhibiting hepatitis B virus (hepatitis B virus, HBV) gene expression, a pharmaceutical composition and a conjugate containing the siRNA as an active ingredient, and the siRNA, the pharmaceutical composition and the siRNA Use of the conjugate in preparing a medicament for treating and/or preventing hepatitis B.
  • hepatitis B virus hepatitis B virus
  • Viral hepatitis B (also known as hepatitis B or hepatitis B) is a class of infectious diseases that seriously threaten the world, especially China.
  • the two major types of hepatitis B prevention and treatment drugs recognized globally are interferon and nucleoside analogues, but these two types of drugs have many disadvantages such as easy drug resistance or limited use after use. For example, interferon is prone to adverse reactions, nucleoside drugs have drug resistance and recurrence after drug withdrawal.
  • small interfering RNA small interfering RNA
  • siRNA can inhibit or block any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), e.g. Disease gene) expression, so as to achieve the purpose of treating the disease.
  • RNAi RNA interference
  • siRNA on the other hand, in the pharmaceutical research of siRNA, off-target effect is one of the important side effects related to toxicity.
  • many siRNAs that have shown excellent pharmaceutical activity in preclinical pharmaceutical studies are difficult to be used in actual drug development due to the toxicity caused by their off-target effects.
  • the drug siRNA expected to be used in the preparation of reagents for patients should undoubtedly have low toxicity, including low toxicity caused by off-target effects. Therefore, how to obtain siRNAs with low off-target effects still needs to be further explored in this field.
  • PCT International Application WO2016077321A1 discloses a number of siRNAs specifically targeting the X gene region of the HBV gene and methods for their delivery, and the stability of the siRNAs in plasma is enhanced by modifying the nucleotides of the siRNAs.
  • siRNA modification schemes In view of the importance of modification schemes to siRNA, it is still necessary to find new siRNA modification schemes to further enhance the stability of siRNA and maintain its activity.
  • siRNAs with stabilizing modified nucleotides at specific positions in the sequence showed no stabilizing modifications compared to corresponding positions
  • the present disclosure provides a siRNA, the siRNA comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and 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 Nucleotide, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, and the nucleotide sequence II is at least partially reversely complementary to the first nucleotide sequence , the first nucleotide sequence is a 19-nucleotide nucleotide sequence in the mRNA expressed by the HBV gene, according to the direction from the 5' end to the 3' end, the nucleotide sequence II At least
  • the present disclosure also provides an siRNA, the siRNA comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the Both the nucleotide sequence I and the nucleotide sequence II consist of 19 nucleotides, and each nucleotide in the nucleotide sequence I and the nucleotide sequence II is independently modified or unmodified Modified nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partly reverse complementary to form a double-stranded region, and the nucleotide sequence II is at least partly connected to the first nucleotide sequence Reverse complementary, the first nucleotide sequence is a 19-nucleotide nucleotide sequence in the mRNA expressed by the HBV gene, according to the direction from the 5' end to the 3' end, the nucleoside At least one of the 3-6
  • the present disclosure also provides an siRNA conjugate comprising the siRNA provided in the present disclosure and a conjugation group linked 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 linked covalently or non-covalently, and each targeting group is selected from Ligands for surface receptor binding.
  • the present disclosure also provides a pharmaceutical composition, which contains the siRNA provided in the present disclosure, and/or the conjugate provided in the present disclosure and a pharmaceutically acceptable carrier.
  • 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 in preparation for treatment and/or prevention of mRNA levels related to HBV gene expression Use in medicines for related diseases or symptoms.
  • the present disclosure also provides a method for treating and/or preventing diseases or symptoms related to the mRNA level of HBV gene expression, the method comprising administering the siRNA of the present disclosure to a subject in need thereof, and /or the pharmaceutical composition of the present disclosure and/or the siRNA conjugate of the present disclosure.
  • the present disclosure also provides a method for inhibiting the expression level of HBV genes in cells, the method comprising administering an effective dose of the siRNA of the present disclosure, and/or the pharmaceutical composition of the present disclosure and/or the siRNA of the present disclosure
  • the siRNA conjugate is contacted with the cells.
  • 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 good stability and low off-target effect, and has good inhibitory activity of HBV gene expression, which is specifically described as follows.
  • the siRNAs, pharmaceutical compositions and/or siRNA conjugates of the present disclosure may have less off-target effects and/or toxic reactions due to off-target effects in vitro.
  • the siRNA conjugate provided by the present disclosure when the administration concentration of siRNA is 40nM, and the sequence complementary to the siRNA antisense strand in the conjugate to be tested is amplified 5 times, the siRNA conjugate is still not detected.
  • the conjugates of the present disclosure showed a more significant reduction in off-target effects than reference conjugates that did not contain stabilizing modified nucleotides.
  • the liver weight hardly increased, and showed significantly lower toxicity than the reference siRNA conjugate in terms of hepatic steatosis and inflammation .
  • the siRNA conjugates provided by the present disclosure have an in vitro sicheck system with an inhibition rate of off-target target sequences not higher than 25%, which is significantly higher than that of reference siRNA conjugates that do not contain stabilized modified nucleotides. Lower off-target effects.
  • mice given the siRNA conjugate of the present disclosure at a dose of 30 mg/kg the toxicity in hepatic steatosis was shown to be close to that of the blank control group, and it was significantly lower than that given to mice not containing stabilized modified nucleosides.
  • Mice with the reference conjugate of acid showed hepatic steatosis.
  • the mice administered with the siRNA conjugate of the present disclosure also showed a response close to that of the blank control group in terms of hepatic steatosis and inflammation, without significant abnormalities, indicating that the siRNA conjugate of the present disclosure has very low hepatotoxicity.
  • the siRNA conjugates of the present disclosure not only have comparable or significantly higher inhibitory activity on the target target sequence, but also show significantly higher inhibitory activity. Low off-target effects.
  • the blood biochemical indexes in the mice given the siRNA conjugate of the present disclosure at a dose of 30 mg/kg were significantly reduced, and remained close to the level of the blank control group; And it showed significantly lower toxicity in histopathology, no abnormalities were found in most mice, and only some mice showed mild hepatocyte degeneration.
  • the siRNA, pharmaceutical composition and/or siRNA conjugates of the present disclosure exhibit excellent HBV gene regulation activity in vitro experiments.
  • the conjugates provided by the present disclosure have good in vitro inhibitory activity at a concentration of 10 nM, especially conjugate 2 and conjugate 4, the inhibitory activity of which is as high as 99.4%. Compared with the reference conjugate of acid, it can show substantially the same or even higher activity.
  • the siRNA conjugate of the present disclosure exhibits excellent HBV mRNA inhibitory activity in 44Bri mouse primary liver cells. At 10 nM siRNA concentration, the HBV mRNA inhibitory rate is at least 81.29%, and the highest can reach 93.06%.
  • the conjugates of the disclosure all showed comparable target Sequence inhibitory activity, for example, the conjugates of the present disclosure have lower IC50 /(GSCM) values than the reference conjugates, the IC50/(GSCM) values are reduced by between 0.0137-0.0261nM, compared to the reference conjugates The IC50/(GSCM) values of the conjugates were reduced by 29.5%-56.3% compared to the conjugates.
  • conjugates of the disclosure all exhibited greater IC 50 /(MOS-5) /IC 50 /(GSCM) values compared to reference conjugates that did not contain stabilizing modified nucleotides, especially Conjugate 2, IC 50 /(MOS-5) /IC 50 /(GSCM) value is 3.4 times that of reference conjugate 1.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure have significantly lower off-target effects and toxic reactions caused by off-target effects, and can also effectively inhibit the expression of HBV genes in vitro, so they can be used in a significant While having higher safety, it can effectively treat and/or prevent diseases or symptoms related to the level of mRNA expressed by the HBV gene, and has a good application prospect.
  • Figure 1 is a bar graph of the target sequence inhibitory activity of the conjugate of this example and the reference conjugate at a concentration of 10 nM in mouse liver primary cells.
  • Figure 2 is a scatter diagram of the relative expression level of HBV mRNA in the liver of 44Bri mice after administration of the conjugate of this embodiment or the reference conjugate and PBS, respectively.
  • Figure 3A and Figure 3B are scatter diagrams of the concentrations of ALT and AST in mouse serum after administration of the conjugate of this example or the reference conjugate and PBS, respectively.
  • Fig. 4 is a histogram of the relative expression level of HBV mRNA in 44Bri mouse liver primary cells after free intake of the siRNA conjugate of the present disclosure or the reference siRNA conjugate and the reference conjugate NC.
  • Figure 5 is a scatter plot of the relative expression level of HBV mRNA in the liver of 44Bri mice given the siRNA conjugate of the present disclosure or a reference siRNA conjugate and PBS.
  • FIG. 6 is a bar graph of the relative expression level of HBV mRNA in 44Bri mouse liver primary cells after freely ingesting the siRNA conjugate of the present disclosure or the reference siRNA conjugate and the reference conjugate NC.
  • HBV mRNA refers to the mRNA having the sequence shown in Genbank registration number NC_003977.1
  • HBV gene refers to the gene that transcribes the above-mentioned HBV mRNA.
  • the uppercase letters C, G, U, and A represent the base composition of nucleotides;
  • the lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is methoxy Modified nucleotides;
  • the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that between the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate subunit connection;
  • P1 indicates that the adjacent nucleotide on the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, in some embodiments, P1 It is VP, Ps or P that represents a specific modification, wherein the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is vinyl phosphate (5'-(E)-
  • fluorinated modified nucleotide refers to the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with fluorine
  • non-fluorinated modified nucleotide refers to Nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribose group of a nucleotide with a non-fluorine group.
  • Nucleotide analog means a nucleic acid capable of replacing nucleotides, but is structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymus The group of pyrimidine deoxyribonucleotides. Such as isonucleotides, bridged nucleic acid (BNA) or acyclic nucleotides.
  • BNA bridged nucleic acid
  • methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • the reverse complementarity means that in a double-stranded nucleic acid molecule, the bases of one strand each pair with the bases of the other strand in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) is paired.
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary form.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” means that there is no more than one base mismatch between two nucleotide sequences; “complete reverse complement” means that there is no base mismatch between two nucleotide sequences.
  • nucleoside monomer refers to the The type and sequence of nucleotides in siRNA or siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites also known as Nucleoside phosphoramidites) used in solid-phase synthesis of 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.
  • alkyl refers to straight and branched chains having the specified number of carbon atoms, typically 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms.
  • C1-C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residue having a specific number of carbons all branched and straight chain forms having that number of carbons are intended to be encompassed; thus, for example, "butyl” is meant to include n-butyl, sec-butyl , isobutyl and tert-butyl; “propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to the same residues as alkyl but with two points of attachment.
  • alkenyl means an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double Obtained by removing a molecule of hydrogen.
  • the group can be in the cis or trans configuration of the double bond.
  • Typical alkenyl groups include, but are not limited to: vinyl; propenyl such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1- group, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl and the like.
  • alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residues as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond formed through adjacent carbon atoms of the parent alkyl group. Obtained by removing two molecules of hydrogen.
  • Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.
  • alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to the same residues as alkynyl but with two points of attachment.
  • alkoxy refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentyloxy, etc.
  • An alkoxy group typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbons of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e. contains cyclic rings according to the Hückel theory , Delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl.
  • Arylene is a subset of aryl and refers to the same residues as aryl but with two points of attachment.
  • Heteroaryl refers to a radical derived from a 3- to 18-membered aromatic ring containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., contains cyclic delocalization according to Hückel's theory (4n +2) ⁇ -electron system.
  • Heteroaryl includes fused or bridged ring systems.
  • the heteroatoms in the heteroaryl are oxidized heteroatoms.
  • one or more nitrogen atoms are included in the heteroaryl group. In some embodiments, one or more of the nitrogen atoms in the heteroaryl group are quaternized nitrogen atoms.
  • a heteroaryl is attached to the rest of the molecule through any ring atom.
  • heteroaryl groups include, but are not limited to: azepanyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzobisoxazolyl, benzofuranyl, benzene oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl (benzo[b][1,4]dioxepinyl), benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuryl, benzofuranone , benzothienyl, benzo
  • hydroxy protecting groups can be used in the present disclosure.
  • protecting groups render a chemical functionality insensitive to specific reaction conditions and can be added and removed at that functionality in a molecule without substantially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, cited in Each of the above-mentioned documents is incorporated herein in its entirety.
  • protecting groups are stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups useful herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanth-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups useful herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4"-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or a marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and any species poultry.
  • treatment refers to means of obtaining a beneficial or desired result, including but not limited to therapeutic benefit.
  • Treatment means eradicating or ameliorating the underlying disorder being treated.
  • therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, whereby improvement is observed in the subject, although the subject may still be afflicted by the underlying disorder.
  • prevention refers to means of obtaining a beneficial or desired result, including but not limited to prophylactic benefit.
  • prophylactic benefit the double-stranded siRNA, pharmaceutical composition or siRNA conjugate may be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, A diagnosis of the disease has not yet been made, even if possible.
  • the present disclosure provides an siRNA with high HBV gene suppression activity and low off-target effect.
  • the siRNA of the present disclosure comprises a sense strand and an antisense strand
  • the sense strand comprises a nucleotide sequence I
  • the antisense strand comprises a 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 independently a modified or unmodified nucleotide, and the nucleotide sequence I and the nucleotide sequence II is at least partially reverse-complementary to form a double-stranded region, the nucleotide sequence II is at least partially reverse-complementary to the first segment of the nucleotide sequence, and the first segment of the nucleotide sequence It is a 19-nucleotide nucleotide sequence in the mRNA expressed by the HBV gene, according to the direction from the 5' end to the 3' end, in the 3rd to 6
  • the third or fifth nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.
  • no more than 2 nucleotides in the 3rd to 9th nucleotides in the nucleotide sequence II are the stabilizing modified nucleosides acid.
  • the third and/or fifth 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.
  • all 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 stabilizingly modified nucleotide, it also contains stabilizing modified nucleotides other than the 3rd to 9th nucleotides , may significantly affect the ability of the siRNA to regulate the expression level of the target sequence.
  • “increased thermal stability of siRNA” in the context of the present disclosure refers to an increase in the double-strand thermal dissociation temperature (Tm) of said siRNA.
  • “increased thermal stability of siRNA” refers to an increase in the Tm of the siRNA by at least 0.05°C, and in some embodiments refers to an increase in the Tm of the siRNA by 0.1-6°C. In some embodiments, the Tm of the siRNA is increased by 0.5-4°C.
  • the antisense strand in the siRNA of the present disclosure has substantially no effect on the binding ability of the mRNA expressed by the HBV gene, while the binding ability between the off-target target mRNA is significantly reduce, thereby reducing or even eliminating off-target effects.
  • each of the stabilizing modification groups independently has a structure represented by -XR, wherein 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, 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, substituted C 6 -C 8 aryl, the substituted C 2 -C 6 alkyl, substituted C 6 -C 8 Aryl or substituted C 1 -C 6 alkyl means that one or more hydrogen atoms in C 2 -C 6 alkyl, C 6 -C 8 aryl or C 1 -C 6 alkyl are replaced by substituents and The group formed, the substituent is selected from one or more of the following substituents: C 1 -C 3 alkyl,
  • each of said stabilizing modification groups is independently selected from 2'-O-methoxyethyl, 2'-O-allyl, 2'-allyl, 2'-O -2-N-methylamino-2-oxyethylene ethyl, 2'-O-2-N,N-dimethylaminoethyl, 2'-O-3-aminopropyl and 2'-O One of -2,4-dinitrophenyl.
  • each of said stabilizing modification groups is 2'-O-methoxyethyl.
  • the present disclosure also provides an siRNA, the siRNA comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the Both the nucleotide sequence I and the nucleotide sequence II consist of 19 nucleotides, and each nucleotide in the nucleotide sequence I and the nucleotide sequence II is independently modified or unmodified Modified nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partly reverse complementary to form a double-stranded region, and the nucleotide sequence II is at least partly connected to the first nucleotide sequence Reverse complementary, the first nucleotide sequence is a 19-nucleotide nucleotide sequence in the mRNA expressed by the HBV gene, according to the direction from the 5' end to the 3' end, the nucleoside At least one of the 3-6
  • the third or fifth nucleotide in the nucleotide sequence II is 2'-O-methoxy Ethyl-modified nucleotides. In some embodiments, according to the direction from the 5' end to the 3' end, no more than 2 nucleotides in the 3rd to 9th nucleotides in the nucleotide sequence II are 2'-O-methoxy Ethyl-modified nucleotides.
  • the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II if not 2'-O-methoxyethyl Modified nucleotides are 2'-fluoro-modified nucleotides.
  • all nucleotides in the nucleotide sequence II are modified nucleotides; according to 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 2'-O-methoxyethyl modified nucleotides, 2'-fluoro-modified nucleotides, the other in said nucleotide sequence II
  • the nucleotides are each independently one of non-fluorinated modified nucleotides.
  • the siRNA of the present disclosure may be the following first and/or second and/or third and/or fourth siRNA, and each siRNA is described below.
  • the siRNA of the disclosure is a first siRNA.
  • the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and there are no more than 3 nucleotide differences, and the nucleotide sequence II is identical to the nucleotide sequence shown in SEQ ID NO: 2
  • the nucleotide sequences shown are of equal length and differ by no more than 3 nucleotides:
  • said Z 1 is A
  • Z 2 is U
  • said nucleotide sequence I contains a nucleotide Z 3 corresponding to Z 1
  • said nucleotide sequence II contains a nucleotide Z 3 corresponding to Z 2
  • Nucleotide Z4 said Z4 being the first nucleotide at the 5' end of said antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:1.
  • corresponding position refers to the same position in the nucleotide sequence from the same end of the nucleotide sequence, for example, the first nucleus at the 3' end of the nucleotide sequence I
  • a nucleotide is a nucleotide at a position corresponding to the first nucleotide of SEQ ID NO:1.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 may include the difference at the Z3 position and/or any other core in the nucleotide sequence I Nucleotide differences at nucleotide positions.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 may include the Z3 position and/or the core adjacent to Z3 Nucleotide differences at nucleotide positions.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 includes a difference at the Z4 position, and Z4 is selected from A, G or C.
  • the Z3 is a nucleotide complementary to Z4 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 is the difference at the Z4 position, and Z4 is selected from A, G or C.
  • each U in the nucleotide sequence of the disclosed siRNA can be optionally replaced by T.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two core There is no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse Toward complementarity means that there are no mismatches between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II and the nucleotides at positions 1-17 of the first nucleotide sequence Nucleotides are perfectly reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I. Or according to the direction from the 5' end to the 3' end, the second nucleotide in the nucleotide sequence II and according to the direction from the 3' end to the 5' end, the second nucleotide in the nucleotide sequence I base mismatch between nucleotides. By including the base mismatch, the target gene expression inhibitory activity of the siRNA of the present disclosure can be further improved while maintaining a low off-target effect.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, the nucleotide Sequence II is the nucleotide sequence shown in SEQ ID NO:7:
  • Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3 .
  • Z3 is A and Z4 is U.
  • 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 antisense strand provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/ 25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand and the antisense strand of the siRNA is 19/21, 21, 21, 21, 21/
  • the sense strand further comprises a nucleotide sequence III
  • the antisense strand further comprises a nucleotide sequence IV
  • each nucleotide of the nucleotide sequence III is independently non-fluorinated
  • the length of the nucleotide sequence III is 1, 2, 3 or 4 nucleotides, the length of the nucleotide sequence IV and the nucleotide sequence III are equal, and the nucleotide sequence IV It is substantially reverse complementary or fully reverse complementary to the nucleotide sequence III, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence The 3' end of the nucleotide sequence II,
  • the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1, and the difference is no more than 3 nucleotides, and the nucleotide sequence III and IV are both 1 nucleotide in length, the base of the nucleotide sequence III is A, the base of the nucleotide sequence IV 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 lengths of the nucleotide sequences III and IV are both 2 nucleotides, and the base composition of the nucleotide sequence III is is UA, the base composition of the nucleotide sequence IV is UA, the base composition of the second nucleotide sequence is UA, and at this time, the length ratio of the sense strand and the antisense strand is 21/21; Alternatively, the lengths of the nucleotide sequences III and IV are both 1 nu
  • 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 up.
  • the siRNA of the present disclosure is the second RNA, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:46, and has no more than 3 nucleotide differences , and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 47, and there are no more than 3 nucleotide differences:
  • the nucleotide sequence I contains a nucleotide Z 7 corresponding to Z 5
  • the nucleotide sequence II contains a nucleotide corresponding to Z 6 Z 8 , said Z 8 being the first nucleotide at the 5' end of said antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:46.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:46 there is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:47.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:46 may include the difference at the Z 7 position and/or any other core in the nucleotide sequence I Nucleotide differences at nucleotide positions.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:46 may include the Z 7 position and/or the core adjacent to the Z 7 Nucleotide differences at nucleotide positions.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:47 includes a difference at the Z 8 position, and Z 8 is selected from U, G or C.
  • the Z7 is a nucleotide complementary to Z8 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:47 is the difference at the Z 8 position, and Z 8 is selected from U, G or C.
  • each U in the nucleotide sequence of the disclosed siRNA can be optionally replaced by T.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two core There is no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse Toward complementarity means that there are no mismatches between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II and the nucleotides at positions 1-17 of the first nucleotide sequence Nucleotides are perfectly reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I. Or according to the direction from the 5' end to the 3' end, the second nucleotide in the nucleotide sequence II and according to the direction from the 3' end to the 5' end, the second nucleotide in the nucleotide sequence I base mismatch between nucleotides. By including the base mismatch, the target gene expression inhibitory activity of the siRNA of the present disclosure can be further improved while maintaining a low off-target effect.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51
  • nucleotide sequence II Is the nucleotide sequence shown in SEQ ID NO:52:
  • Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7 .
  • Z7 is U and Z8 is A.
  • the length of the sense strand and the antisense strand is the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides, thus, the present disclosure
  • the length ratio of the siRNA sense strand and antisense strand provided can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/ 26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand and the antisense strand of the siRNA is 19/21, 21/23 or 23/
  • the sense strand further comprises a nucleotide sequence III
  • the antisense strand further comprises a nucleotide sequence IV
  • each nucleotide of the nucleotide sequence III is independently non-fluorinated
  • the length of the nucleotide sequence III is 1, 2, 3 or 4 nucleotides, the length of the nucleotide sequence IV and the nucleotide sequence III are equal, and the nucleotide sequence IV It is substantially reverse complementary or fully reverse complementary to the nucleotide sequence III, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence The 3' end of the nucleotide sequence II,
  • the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:46, and the difference is no more than 3 nucleotides, and the nucleotide sequence III
  • the length of the nucleotide sequence IV and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, the base of the nucleotide sequence IV is A, and the second nucleotide The base of the sequence is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides, so
  • the base composition of the nucleotide sequence III is UU
  • the base composition of the nucleotide sequence IV is AA
  • the base composition of the second nucleotide sequence is UU
  • the length ratio of the sense strand is 21/21; or, the lengths of
  • 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 the third siRNA, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:91, and has no more than 3 nucleotide differences , and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:92, and there are no more than 3 nucleotide differences:
  • the nucleotide sequence I contains a nucleotide Z 11 corresponding to Z 9
  • the nucleotide sequence II contains a nucleotide corresponding to Z 10 Z 12 , said Z 12 being the first nucleotide at the 5' end of said antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:91.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:91 there is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:92.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:91 may include the difference at the Z11 position and/or any other core in the nucleotide sequence I Nucleotide differences at nucleotide positions.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 91 may include the Z11 position and/or the core adjacent to Z11 Nucleotide differences at nucleotide positions.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:92 includes a difference at Z12 position, and Z12 is selected from A, G or C.
  • the Z 11 is a nucleotide complementary to Z 12 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:92 is the difference at Z12 position, and Z12 is selected from A, G or C.
  • each U in the nucleotide sequence of the disclosed siRNA can be optionally replaced by T.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two core There is no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse Toward complementarity means that there are no mismatches between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II and the nucleotides at positions 1-17 of the first nucleotide sequence Nucleotides are perfectly reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I. Or according to the direction from the 5' end to the 3' end, the second nucleotide in the nucleotide sequence II and according to the direction from the 3' end to the 5' end, the second nucleotide in the nucleotide sequence I base mismatch between nucleotides. By including the base mismatch, the target gene expression inhibitory activity of the siRNA of the present disclosure can be further improved while maintaining a low off-target effect.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:97:
  • Z11 is selected from A, U, G or C, and Z12 is a nucleotide complementary to Z11 .
  • Z 11 is A and Z 12 is U.
  • the length of the sense strand and the antisense strand is the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides, thus, the present disclosure
  • the length ratio of the siRNA sense strand and antisense strand provided can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/ 26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand and the antisense strand of the siRNA is 19/21, 21/23 or 23/
  • the sense strand further comprises a nucleotide sequence III
  • the antisense strand further comprises a nucleotide sequence IV
  • each nucleotide of the nucleotide sequence III is independently non-fluorinated
  • the length of the nucleotide sequence III is 1, 2, 3 or 4 nucleotides, the length of the nucleotide sequence IV and the nucleotide sequence III are equal, and the nucleotide sequence IV It is substantially reverse complementary or fully reverse complementary to the nucleotide sequence III, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence The 3' end of the nucleotide sequence II,
  • the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:91, and the difference is no more than 3 nucleotides, and the nucleotide sequence III
  • the length of the nucleotide sequence IV and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, the base of the nucleotide sequence IV is U, and the second nucleotide The base of the sequence is A; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides, so
  • the base composition of the nucleotide sequence III is GA
  • the base composition of the nucleotide sequence IV is UC
  • the base composition of the second nucleotide sequence is GA
  • the length ratio of the sense strand is 21/21; or, the lengths of the nu
  • 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 the fourth siRNA, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 136, and has no more than 3 nucleotide differences , and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 137, and there are no more than 3 nucleotide differences:
  • the nucleotide sequence I contains a nucleotide Z 15 corresponding to Z 13
  • the nucleotide sequence II contains a nucleotide corresponding to Z 14 Z 16 , said Z 16 being the first nucleotide at the 5' end of said antisense strand.
  • the first nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:136.
  • the sense strand only comprises nucleotide sequence I
  • the antisense strand comprises only nucleotide sequence II.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 136 may include the difference at the Z 15 position and/or any one of the nucleotide sequence I Nucleotide differences at other nucleotide positions.
  • nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 136 may include the Z 15 position and/or the core adjacent to Z 15 Nucleotide differences at nucleotide positions.
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 137 includes a difference at position Z16 , and Z16 is selected from A, G or C.
  • the Z15 is a nucleotide complementary to Z16 .
  • the difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:137 is the difference at position Z16 , and Z16 is selected from A, G or C.
  • each U in the nucleotide sequence of the disclosed siRNA can be optionally replaced by T.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two core There is no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse Toward complementarity means that there are no mismatches between the two nucleotide sequences.
  • nucleotides at positions 3-19 of the nucleotide sequence II and the nucleotides at positions 1-17 of the first nucleotide sequence Nucleotides are perfectly reverse complementary. In some embodiments, the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I. Or according to the direction from the 5' end to the 3' end, the second nucleotide in the nucleotide sequence II and according to the direction from the 3' end to the 5' end, the second nucleotide in the nucleotide sequence I base mismatch between nucleotides. By including the base mismatch, the target gene expression inhibitory activity of the siRNA of the present disclosure can be further improved while maintaining a low off-target effect.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140 or SEQ ID NO:141
  • nucleotide sequence II Is the nucleotide sequence shown in SEQ ID NO:142:
  • Z15 is selected from A, U, G or C, and Z16 is a nucleotide complementary to Z15 .
  • Z 15 is A and Z 16 is U.
  • the length of the sense strand and the antisense strand is the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides, thus, the present disclosure
  • the length ratio of the siRNA sense strand and antisense strand provided can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/ 26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand and the antisense strand of the siRNA is 19/21, 21/23 or 23/
  • the sense strand further comprises a nucleotide sequence III
  • the antisense strand further comprises a nucleotide sequence IV
  • each nucleotide of the nucleotide sequence III is independently non-fluorinated
  • the length of the nucleotide sequence III is 1, 2, 3 or 4 nucleotides, the length of the nucleotide sequence IV and the nucleotide sequence III are equal, and the nucleotide sequence IV It is substantially reverse complementary or fully reverse complementary to the nucleotide sequence III, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence The 3' end of the nucleotide sequence II,
  • the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 136, and the difference is no more than 3 nucleotides, and the nucleotide sequence III
  • the length of the nucleotide sequence IV and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is C, the base of the nucleotide sequence IV is G, and the second nucleotide The base of the sequence is C; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides, so
  • the base composition of the nucleotide sequence III is GC
  • the base composition of the nucleotide sequence IV is GC
  • the base composition of the second nucleotide sequence is GC
  • the length ratio of the sense strand is 21/21; or, the lengths
  • 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 .
  • nucleotide sequence V nucleotide modifications in siRNA, and modified sequences are applicable to the above-mentioned siRNAs of the present disclosure, such as the first siRNA, the second siRNA, the third siRNA, and the fourth siRNA. That is, if there is no specific description, the following description of siRNA should be regarded as describing the above-mentioned siRNA of the present disclosure, for example, describing the first siRNA, the second siRNA, the third siRNA and the fourth siRNA one by one.
  • the siRNA also contains the nucleotide sequence V".
  • the sense strand and the antisense strand are of different lengths
  • the antisense strand also contains a nucleotide sequence V
  • each nucleotide of the nucleotide sequence V is independently modified by non-fluorine
  • the length of the nucleotide sequence V is 1 to 3 nucleotides, connected at the 3' end of the antisense strand, constituting the 3' overhang of the antisense strand.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25, or 23/26.
  • the length of the nucleotide sequence V is 2 nucleotides, thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/21, 21/23 or 23 /25.
  • each nucleotide in the nucleotide sequence V can be any nucleotide, in order to facilitate synthesis and save cost.
  • the length of the nucleotide sequence V is 2 nucleotides, and according to the direction from the 5' end to the 3' end, the nucleotide sequence V is two consecutive thymine deoxyribose Nucleotide (dTdT), two consecutive uracil ribonucleotides (UU); or, in order to improve the affinity between the siRNA antisense strand and the target mRNA, the nucleotide sequence V is completely reversed to the third nucleotide sequence Complementary to each other, the third nucleotide sequence refers to the mRNA expressed by the HBV gene that is adjacent to the first nucleotide sequence or the second nucleotide sequence and is equal in length to the nucleotide sequence V the nucleotide sequence. Therefore, in some embodiments, the ratio of the lengths of the
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 1, and the base composition of the third nucleotide sequence is It is UA.
  • the positive sense strand of described siRNA contains the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, and described antisense strand contains such as SEQ ID NO: Nucleotide sequence shown in 8:
  • said Z4 is the first nucleotide at the 5' end of the antisense strand
  • Z3 is selected from A, U, G or C
  • Z4 is a nucleotide complementary to Z3 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and the antisense strand of the siRNA contains the following Nucleotide sequence shown in SEQ ID NO:13:
  • said Z4 is the first nucleotide at the 5' end of the antisense strand
  • Z3 is selected from A, U, G or C
  • Z4 is a nucleotide complementary to Z3 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 46, and the base composition of the third nucleotide sequence is It is UU.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51, and the antisense strand of the siRNA contains the sequence shown in SEQ ID Nucleotide sequence shown in NO:53:
  • said Z8 is the first nucleotide at the 5' end of the antisense strand
  • Z7 is selected from A, U, G or C
  • Z8 is a nucleotide complementary to Z7 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57, and the antisense strand of the siRNA contains the following The nucleotide sequence shown in SEQ ID NO:58:
  • said Z8 is the first nucleotide at the 5' end of the antisense strand
  • Z7 is selected from A, U, G or C
  • Z8 is a nucleotide complementary to Z7 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 91, and the base composition of the third nucleotide sequence is It is GA.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96, and the antisense strand of the siRNA contains the sequence shown in SEQ ID Nucleotide sequence shown in NO:98:
  • said Z 12 is the first nucleotide at the 5' end of the antisense strand
  • Z 11 is selected from A, U, G or C
  • Z 12 is a nucleotide complementary to Z 11 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102
  • the antisense strand of the siRNA contains the following Nucleotide sequence shown in SEQ ID NO:103:
  • said Z 12 is the first nucleotide at the 5' end of the antisense strand
  • Z 11 is selected from A, U, G or C
  • Z 12 is a nucleotide complementary to Z 11 .
  • the first nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 136
  • the base composition of the third nucleotide sequence is is the GC.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140 or SEQ ID NO:141
  • the antisense strand of the siRNA contains the sequence shown in SEQ ID Nucleotide sequence shown in NO:143:
  • said Z 16 is the first nucleotide at the 5' end of the antisense strand, Z 15 is selected from A, U, G or C, and Z 16 is a nucleotide complementary to Z 15 ;
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146 or SEQ ID NO:147, and the antisense strand of the siRNA contains the following Nucleotide sequence shown in SEQ ID NO:148:
  • said Z 16 is the first nucleotide at the 5' end of the antisense strand
  • Z 15 is selected from A, U, G or C
  • Z 16 is a nucleotide complementary to Z 15 .
  • nucleotides in the siRNAs 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 nucleotides, and these modifications on the nucleotide groups will not cause the function of the siRNA of the present disclosure to inhibit HBV gene expression to be significantly weakened or lost.
  • modified nucleotide refers to a nucleotide or nucleotide analog, or a nucleoside, in which the 2' hydroxyl group of the ribose group of a nucleotide is substituted by other groups.
  • the base on the acid is the nucleotide of the modified base.
  • the modified nucleotides will not cause obvious weakening or loss of the function of siRNA to inhibit gene expression.
  • modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected.
  • the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II if not the stabilizing modified nucleotides, 2'-fluoro-modified nucleotides.
  • all the nucleotides in the nucleotide sequence II are modified nucleotides; according to the direction from the 5' end to the 3' end, all The 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II, if not the stabilized modified nucleotides, are 2'-fluoro-modified nucleotides, and the nucleotide sequence
  • the other nucleotides in II are each independently one of non-fluorinated modified nucleotides.
  • the 7th-9th nucleotides of the nucleotide sequence I are 2'-fluoro-modified nucleotides. In some embodiments, all the nucleotides in the nucleotide sequence I are modified nucleotides; according to the direction from the 5' end to the 3' end, the 7th-9th nucleotides in the nucleotide sequence I Each nucleotide is a 2'-fluorinated modified nucleotide, and the other nucleotides in the nucleotide sequence I are each independently one of non-fluorinated modified nucleotides.
  • the siRNA of the present disclosure can achieve a good balance between gene expression regulation activity and in vivo stability by having the above-mentioned modification.
  • fluorine-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, which has the structure shown in the following formula (7).
  • Non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted by a non-fluorine group A sort of.
  • Nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'- Alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-deoxynucleosides One of the glycosides.
  • the 2'-alkoxy-modified nucleotides are methoxy-modified nucleotides (2'-OMe), as shown in formula (8).
  • the 2'-amino modified nucleotide (2'-NH 2 ) is represented by formula (9).
  • the 2'-deoxynucleotide (DNA) is shown in formula (10):
  • Nucleotide analogs are capable of replacing nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymidine ribonucleotides group of ribonucleotides.
  • the nucleotide analogs can be isonucleotides, bridged nucleic acid (BNA for short) or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNAs may contain five-membered, six-membered, or seven-membered ring bridge structures with "fixed" C3'-endosugar constrictions. Typically the bridge is incorporated at the 2'-,4'-position of the ribose to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA etc., wherein, LNA is as shown in formula (12), ENA is as shown in formula (13), and cET BNA is as shown in formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is shown in formula (15), and GNA is shown in formula (16):
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotide refers to a compound formed by changing the position of the base in the nucleotide on the ribose ring.
  • the isonucleotide can be a compound formed by moving a base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18).
  • Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA and GNA.
  • each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, above and below, the methoxy-modified nucleotide refers to the 2' ribose group - Nucleotides in which a hydroxyl group is substituted by a methoxy group.
  • fluoro-modified nucleotide refers to a compound with the structure shown in formula (7) formed by replacing the 2'-hydroxyl group of the nucleotide with fluorine;
  • methoxy-modified nucleotide refers to a compound with the structure shown in formula (8) formed by substituting the 2'-hydroxyl group of the ribose group of a nucleotide with a methoxy group.
  • no more than three non-fluorinated modified nucleotides in the antisense strand are 2'-deoxynucleotides, and each of the remaining non-fluorinated modified nucleotides is formazan Oxygen-modified nucleotides; or, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide; the methoxy-modified nucleotide means that the 2'-hydroxyl of the ribose group is Nucleotides substituted with methoxy groups.
  • the siRNA comprising stabilizing modified nucleotides of the present disclosure is an siRNA with the following modifications: in the sense strand, the nucleotide sequence I
  • the 7th, 8th, and 9th or 5th, 7th, 8th, and 9th nucleotides are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleosides acid; according to the direction from the 5' end to the 3' end, in the antisense strand, the 2nd, 6th, 14th, 16th or 2nd, 6th, 8th, 9th, 14th, or 2nd, 6th, 8th, 9th, 14th,
  • the 16th nucleotide is a fluorine-modified nucleotide
  • the 3rd or 5th nucleotide in the antisense strand is a stabilizing modified nucleotide
  • the 18th nucleotide is a 2' - de
  • the siRNA with the above modification is not only low in cost, but also makes it difficult for ribonuclease in the blood to cut nucleic acid, thereby increasing the stability of nucleic acid and making the nucleic acid more resistant to ribonuclease hydrolysis.
  • the above modification reduces the off-target effect of siRNA without significantly reducing the inhibitory performance of siRNA.
  • the siRNA provided by the present disclosure is siHBVa1-M1, siHBVa1-M2, siHBVa2-M1, siHBVa2-M2, siHBVa3-M1, siHBVa3-M2, siHBVa4-M1, siHBVa4- M2, siHBVa5-M1, siHBVa5-M2, siHBVa6-M1, siHBVa6-M2, siHBVa7-M1, siHBVa7-M2, siHBVa8-M1, siHBVa8-M2, siHBVb1-M1, siHBVb1-M2, siHBVb2-M1, siHBVb2-M2 , siHBVb3-M1, siHBVb3-M2, siHBVb4-M1, siHBVb4-M2, siHBVb5-M1, siHBVb5-M2, siHBVb6-M1, siHBVb6-M2, siHBVb7-M1, siHBVb7-M2, siHBVa1-M
  • the phosphate group with the modification group is a phosphorothioate subunit formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, The phosphate group with the modification group is a phosphorothioate subunit having a structure as shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.
  • the phosphorothioate subunit linkage is present at at least one of the following positions: the first and the second of either end of the sense strand or the antisense strand between the nucleotides; between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above.
  • phosphorothioate subunit linkages are present at all of the above positions except the 5' end of the sense strand.
  • phosphorothioate subunit linkages are present at all of the above positions except the 3' end of the sense strand.
  • the phosphorothioate subunit linkage is present in at least one of the following positions:
  • the siRNA of the present disclosure is siHBVa1-M1S, siHBVa1-M2S, siHBVa2-M1S, siHBVa2-M2S, siHBVa3-M1S, siHBVa3-M2S, siHBVa4-M1S, siHBVa4-M2S listed in Tables 1a-1d , siHBVa5-M1S, siHBVa5-M2S, siHBVa6-M1S, siHBVa6-M2S, siHBVa7-M1S, siHBVa7-M2S, siHBVa8-M1S, siHBVa8-M2S, siHBVb1-M1S, siHBVb1-M2S, siHBVb2-M1S , siHBVb2-M2S, siHBVb3 -M1S, siHBVb3-M2S, siHBVb4-M1S, siHBVb4
  • the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides or 5'-phosphate analog modified nucleotides are well known to those skilled in the art, for example, 5'-phosphate nucleotides may have the following structure:
  • R is selected from H, OH, methoxy, fluorine;
  • Base represents a nucleic acid base, selected from A, U, C, G or T.
  • the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification shown in formula (2)
  • the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate ( 5'-(E)-vinylphosphonate, E-VP) modified nucleotides, as shown in formula (3), or phosphorothioate modified nucleotides, as shown in formula (5).
  • the siRNAs of the present disclosure are siHBVa1-M1P1, siHBVa1-M2P1, siHBVa2-M1P1, siHBVa2-M2P1, siHBVa3-M1P1, siHBVa3-M2P1, siHBVa4-M1P1, siHBVa4- M2P1, siHBVa5-M1P1, siHBVa5-M2P1, siHBVa6-M1P1, siHBVa6-M2P1, siHBVa7-M1P1, siHBVa7-M2P1, siHBVa8-M1P1, siHBVa8-M2P1, siHBVa1-M1SP1, siHBVa1-M2 SP1, siHBVa2-M1SP1, siHBVa2-M2SP1, siHBVa3-M1SP1, siHBVa3-M2SP1, siHBVa4-M1SP1, siHBVa4-M1SP1, siHBVa5-M2P
  • the inventors of the present disclosure unexpectedly found that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosome stability, significantly low off-target effects, but also retains high gene inhibitory activity.
  • the siRNA provided in the present disclosure can be obtained by conventional siRNA preparation methods in the art (such as solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis has commercialized customized services.
  • a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a correspondingly modified nucleoside monomer, a method for preparing a correspondingly modified nucleoside monomer and introducing a modified nucleotide group Methods of siRNA are also well known to those skilled in the art.
  • the present disclosure provides an siRNA conjugate comprising the siRNA provided by the present disclosure, and a conjugation group conjugated to the siRNA.
  • the conjugation group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the siRNA, the linker and the targeting group or the The delivery auxiliary groups are sequentially connected covalently or non-covalently, each targeting group is selected from ligands capable of binding to cell surface receptors, and each delivery auxiliary group is selected from ligands capable of increasing the siRNA conjugate. biocompatibility of the compound in the delivery target organ or tissue.
  • conjugate means that two or more chemical moieties each having a specific function are covalently linked to each other; accordingly, a “conjugate” is Refers to the compound formed by covalent linkage between the various chemical moieties.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugates should be understood as a general term of multiple siRNA conjugates or siRNA conjugates represented by a certain chemical formula according to the context.
  • conjuggating group should be understood as a specific compound that can be conjugated to siRNA through a reaction, finally forming the siRNA conjugate of the present disclosure.
  • the conjugating group comprises at least one targeting group that is pharmaceutically acceptable and optionally a linker, and the siRNA, the linker and the targeting group are linked sequentially.
  • the siRNA molecule may be non-covalently or covalently conjugated to the conjugating group, for example may be covalently conjugated to the conjugating group.
  • the conjugation site between the siRNA and the conjugating group can be at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site between the siRNA and the conjugation group is at the 3' end of the sense strand of the siRNA.
  • the conjugate group can be attached to the phosphate group, the 2'-position hydroxyl group or the base of the nucleotide. In some embodiments, the conjugate group can also be connected to the hydroxyl group at the 3'-position, at this time, the nucleotides are connected by 2'-5' phosphodiester bonds.
  • the conjugation group is usually connected to the phosphate group of the nucleotide; when the conjugation group is connected to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose sugar ring or base.
  • the targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • suitable linker those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • the types of these linkers, targeting groups and connection methods with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.
  • the targeting group may be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.
  • At least one or each of the targeting groups is selected from ligands capable of binding to cell surface receptors expressing the HBV gene.
  • At least one or each of the targeting groups is selected from ligands capable of binding to mammalian liver parenchymal cell surface receptors (ASGPR).
  • each of said targeting groups is independently a ligand that has an affinity for an asialoglycoprotein receptor on the surface of mammalian liver cells.
  • each of said targeting groups is independently an asialoglycoprotein or a sugar.
  • each of the targeting groups is independently an asialoglycoprotein, such as asialorosomucoid (ASOR) or asialofetuin (ASF).
  • each of the targeting groups is independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D- - Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannose furanose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose , ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D- Galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-galactofuranose, glucosamine, sialic acid
  • the linker in the siRNA conjugates of the present disclosure has a structure as shown in formula (301):
  • k is an integer of 1-3;
  • L A has a structure containing an amide bond as shown in formula (302)
  • L B has a structure containing N-acylpyrrolidine as shown in formula (303), containing carbonyl and oxygen atoms
  • LC is based on hydroxymethyl Linking groups for aminomethane, dimethylolaminomethane or trishydroxymethylaminomethane;
  • n 302 , q 302 and p 302 are each independently an integer of 2-6, optionally, n 302 , q 302 and p 302 are each independently 2 or 3; n 303 is an integer of 4-16, which can be Optionally, n 303 is an integer of 8-12, Indicates the site where the group is covalently attached.
  • each LA is connected to one of the targeting groups through an ether bond, and is connected through the oxygen atom of the hydroxyl group in the L C part and the L C part to form an ether bond;
  • L B is connected through the formula (303)
  • the carbonyl in the formula (303) forms a amide bond with the nitrogen atom of the amino group in the LC part, and forms a phosphate bond or a phosphorothioate bond with the siRNA through the oxygen atom in the formula (303).
  • the siRNA conjugate provided by the present disclosure has a structure as shown in formula (305):
  • Nu represents the siRNA provided by the present disclosure.
  • the linker in the siRNA conjugates of the present disclosure has the structure shown in formula (306):
  • n 306 is an integer of 0-3, and each p 306 is independently an integer of 1-6, Indicates the site where the group is covalently attached; the linking group is connected by an ether bond with the targeting group through the oxygen atom marked by *; the linking group is connected by at least one of the oxygen atoms marked by # One is connected to the siRNA by forming a phosphate bond or a phosphorothioate bond, and the rest are connected with an oxygen atom marked by # to form a hydroxyl group, or to form a C 1 -C 3 alkyl group to form a C 1 -C 3 alkane Oxygen;
  • siRNA conjugates of the present disclosure have a structure as shown in formula (307):
  • Nu represents the siRNA provided by the present disclosure.
  • siRNA conjugates of the present disclosure have the structure shown in formula (308):
  • n1 is an integer selected from 1-3, and 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 are selected from the group consisting of the following groups: C 1 -C 10 alkyl, C 1 -C 10 haloalkane and C 1 -C 10 alkoxy;
  • R 3 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 optional range are the same as above.
  • each M1 is independently selected from one of the ligands that have affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
  • L is defined as a linear alkyl group for convenience, it may not be a linear group or be named differently, such as amine or alkenyl as a result of the substitutions and/or substitutions described above.
  • the length of L is the number of atoms in the chain connecting the two attachment points.
  • a ring obtained by substituting a carbon atom of the linear alkylene group, such as a heterocyclylene or heteroarylene, is counted as one atom.
  • n1 can be an integer of 1-3, and n3 can be an integer of 0-4 , to ensure that the number of M 1 ligands in the conjugate is at least 2; in some embodiments, n1+n3 ⁇ 2, so that the number of M 1 ligands is at least 3, so that the M 1 ligands It is easier to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the entry of the conjugate into cells through endocytosis.
  • n1 is an integer of 1-2
  • n3 is an integer of 0-1
  • n1+n3 2-3.
  • the spatial position between a plurality of M1 ligands can be suitable for 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
  • R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are each independently selected from H, methyl, and ethyl.
  • R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are all H.
  • R 3 is a group with the structure shown in formula A59, wherein E 1 is OH, SH or BH 2 , based on the consideration of the availability of raw materials for preparation, in some embodiments, E 1 is OH or SH.
  • R2 is selected to enable attachment of N on the nitrogen-containing backbone to A59.
  • nitrogen-containing skeleton refers to a chain structure in which carbon atoms connected with R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are connected to N.
  • R2 may be any linking group capable of linking the A59 group to the N on the nitrogen-containing backbone in an appropriate manner.
  • the R2 group needs to contain both the linking site connected to the N on the nitrogen-containing skeleton and the linking site connected to the R3 group. The P-linked junction site.
  • the site connected to N on the nitrogen-containing skeleton in R2 forms an amide bond with N
  • the site connected to P on R3 forms a phosphate bond with P.
  • R is 4-15 atoms in length.
  • R 2 is B5, B6, B5' or B6':
  • the value range of q 2 may be an integer of 1-10, and in some embodiments, q 2 is an integer of 1-5.
  • L1 is 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, L1 is selected from a connection combination of at least 2 of A1, A8, and A10.
  • L can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, the length of L1 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 of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, and in some embodiments, j2 is an integer of 3-5.
  • R' is a C 1 -C 4 alkyl group, and in some embodiments, R' is one of methyl, ethyl and isopropyl.
  • Ra is one of A27, A28, A29, A30 and A31, and in some embodiments, Ra is A27 or A28.
  • Rb is a C 1 -C 5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl and butyl.
  • each of j1, j2, R', Ra, and Rb is selected in formulas A1-A26, so as to realize the connection of the M 1 ligand to the N on the nitrogen-containing backbone, and to make the M 1 ligand
  • the spatial position of is more suitable for the binding of M 1 ligand to the asialoglycoprotein receptor on the liver surface.
  • siRNA conjugates of the present disclosure have the formula (403), (404), (405), (406), (407), (408), (409), (410), (411 ) , (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):
  • Nu represents the siRNA of the present disclosure.
  • P in formula A59 can be connected to any possible position in the siRNA sequence, for example, P in formula A59 can be connected to any nucleotide of the sense strand or antisense strand of siRNA; in some embodiments In the manner, P in the formula A59 is connected to any nucleotide of the sense strand of the siRNA.
  • P in Formula A59 is linked to the end of the sense or antisense strand of the siRNA; in some embodiments, P in Formula A59 is linked to the end of the sense strand of the siRNA.
  • the terminus refers to the first 4 nucleotides counted from one end of the sense strand or the antisense strand.
  • P in Formula A59 is linked to the end of the sense or antisense strand of the siRNA; in some embodiments, P in Formula A59 is linked to the 3' end of the sense strand of the siRNA.
  • a separate antisense strand of siRNA can be released to inhibit target gene expression through RNAi mechanism.
  • P 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 base.
  • P in formula A59 can be linked to the 2' position, 3' position or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond.
  • P 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 P in formula A59 is replaced by a core in the sense strand of siRNA
  • the hydrogen in the 2'-hydroxyl of the nucleotide is attached to the nucleotide, or P in formula A59 is attached to the nucleotide by substituting the hydrogen in the 5'-hydroxyl of the 5' terminal nucleotide of the sense strand of the siRNA.
  • the siRNA comprised by the siRNA conjugates of the present disclosure can be, for example, any of the siRNAs listed in Table 1a, Table 1b, Table 1c, or Table 1d. siRNA conjugates containing these siRNAs exhibited low off-target effects and high mRNA inhibitory activity of HBV gene expression.
  • the uppercase letters C, G, U, and A indicate the base composition of nucleotides;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates The nucleotide adjacent to the left side of the letter f is a fluorinated nucleotide;
  • the lowercase letter d indicates that the nucleotide adjacent to the right side of the letter d is a 2'-deoxynucleotide;
  • the underlined The uppercase letter S indicates that the adjacent nucleotide to the left of the letter S is a stabilizing modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter S are connected by phosphorothioate subunits;
  • P1 indicates The adjacent nucleotide on the right side of P1 is a 5'-phosphate nucleotide
  • S represents a specific stabilizing modification such as moe , wherein the underlined letter combination moe indicates that a nucleotide adjacent to the left side of the letter combination moe has a 2'-O-methoxy Ethyl-modified nucleotides.
  • each S is moe .
  • P1 is VP, Ps or P representing a specific modification
  • the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is vinyl phosphate (5'-(E)- Vinylphosphonate, E-VP) modified nucleotides
  • the letter combination Ps indicates that the adjacent nucleotide on the right side of the letter combination Ps is a phosphorothioate modified nucleotide
  • the capital letter P indicates that the right side of the letter P is the same
  • the adjacent nucleotide is a 5'-phosphate nucleotide.
  • each U in the sequences listed in the above Tables 1a-1d can be arbitrarily replaced by T, which will not significantly affect the activity or off-target effect of the siRNA.
  • the present disclosure provides a pharmaceutical composition, which contains the above-mentioned siRNA, and/or the above-mentioned siRNA conjugate as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (magnetic nanoparticles, such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon nanotubes ( carbon nanotubes), mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), polylysine acid (poly(L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly D Type or L-type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid)copolymer, PLGA), poly(aminoethylethylene phosphate) (poly(2-
  • the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1:( 1-500). In some embodiments, the above weight ratio is 1:(1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds routinely used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent and an osmotic pressure regulator.
  • the pH buffer can be a tris hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5 buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmol/kg (mOsm/kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • the dosage of the preparation made from the pharmaceutical composition will be adjusted due to different administration methods during administration.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation can be used for subcutaneous, intramuscular or intravenous injection administration, but can also be administered to the lungs by spraying, or administered to other organs (such as the liver) through the lungs by spraying, Or deliver the pharmaceutical composition through oropharyngeal inhalation or nasal administration.
  • the pharmaceutical composition is for spray administration.
  • the pharmaceutical composition may be in the form of a liposomal formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipid.
  • the organic amine, helper lipid and pegylated lipid can be selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (which is incorporated herein by reference in its entirety) or its One or more of pharmaceutically acceptable salts or derivatives, helper lipids and pegylated lipids.
  • the organic amine can be a compound represented by formula (201) or a pharmaceutically acceptable salt thereof described in Chinese patent application CN103380113A:
  • X 101 and X 102 are each independently O, S, NA or CA, wherein A is hydrogen or C 1 -C 20 hydrocarbon chain;
  • R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic Group, cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, substituted or unsubstituted, branched or straight chain acyl group, substituted or unsubstituted Substituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;
  • x is an integer of 1-10;
  • R 103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl, the alkyl A radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 can be any of the following formulas (204)-(213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201) Possible points of attachment of the nitrogen atom in , where each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.
  • the organic amine is an organic amine shown in formula (214) and/or an organic amine shown in formula (215):
  • the helper lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives.
  • the pegylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxyl (polyethylene glycol)]-2000.
  • the molar ratio among the organic amine, the helper lipid and the pegylated lipid is (19.7-80):(19.7-80 ):(0.3-50), such as (50-70):(20-40):(3-20).
  • particles of the pharmaceutical composition formed from siRNAs of the disclosure and the amine-containing transfection reagents described above have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm. More typically, the liposome particles have an average diameter of about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the liposome particles have an average diameter of about 30 nm. , 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.
  • the weight of siRNA and total lipid is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1: 18. From about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or from about 1:1: 6 to about 1:10 range.
  • the weight ratio of siRNA to total lipid of the disclosure is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 , 1:14, 1:15, 1:16, 1:17, or 1:18.
  • each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used.
  • the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, only the siRNA provided by the present disclosure can be used to replace the existing siRNA; in some In the embodiment, it can be prepared according to the following method:
  • the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid around room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more of, for example, can be ethanol.
  • the siRNA provided by the present disclosure is dissolved in a buffered saline solution to obtain an aqueous siRNA solution.
  • concentration of the buffered saline solution is 0.05-0.5M, such as 0.1-0.2M
  • the pH of the buffered saline solution is adjusted to 4.0-5.5, such as 5.0-5.2
  • amount of the buffered saline solution is such that the concentration of siRNA does not exceed 0.6mg /mL, for example, can be 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and/or potassium acetate.
  • the lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation.
  • the volume ratio of lipid solution and siRNA aqueous solution is 1:(2-5), for example, it can be 1:4.
  • the incubated liposome preparation Concentrate or dilute the incubated liposome preparation, remove impurities, and sterilize to obtain the pharmaceutical composition provided by the present disclosure, whose physical and chemical parameters are pH 6.5-8, encapsulation efficiency not less than 80%, particle size 40-200nm, polydispersity index not higher than 0.30, osmotic pressure 250-400mOsm/kg; for example, physical and chemical parameters can be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100nm, more The dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution can be performed before, after or simultaneously with the removal of impurities.
  • PBS phosphate buffer saline
  • Various existing methods can be used for the sterilization method, for example, filtration sterilization on a 0.22 ⁇ m filter can be used.
  • siRNA conjugates can be prepared by methods known in the art.
  • WO2015006740A2 describes the preparation method of various siRNA conjugates in detail
  • WO2014025805A1 describes the preparation method of the structure shown in formula (305)
  • Rajeev et al. described the formula (307) in ChemBioChem 2015, 16, 903-908.
  • the method for preparing the structure shown in Chinese patent application CN110959011A discloses in detail the method for preparing the siRNA conjugate shown in formula (308).
  • the contents of the above documents are incorporated herein in their entirety by reference.
  • the siRNA conjugate of the present disclosure can be obtained by other means well known to those skilled in the art.
  • siRNA conjugates of the present disclosure can also be used in combination with other pharmaceutically acceptable adjuvants, which can be one or more of various preparations or compounds routinely used in the art.
  • pharmaceutically acceptable adjuvants can be one or more of various preparations or compounds routinely used in the art.
  • description of the pharmaceutical composition please refer to the above-mentioned information about the present disclosure. Description of the pharmaceutical composition.
  • siRNA composition and siRNA conjugate of the present disclosure
  • the present disclosure provides siRNAs of the present disclosure, and/or pharmaceutical compositions and/or siRNA conjugates prepared for the treatment and/or prevention of diseases or symptoms related to the mRNA level of HBV gene expression Uses in medicine.
  • the disease or symptom associated with the mRNA level of HBV gene expression is at least one of chronic liver disease, inflammation, fibrotic disease, and proliferative disease.
  • the present disclosure provides a method of treating and/or preventing a disease or condition associated with mRNA levels of HBV gene expression, the method comprising administering an siRNA of the present disclosure to a subject in need thereof, and /or the pharmaceutical composition of the present disclosure and/or the siRNA conjugate of the present disclosure.
  • the disease or symptom associated with the mRNA level of HBV gene expression is at least one of chronic liver disease, inflammation, fibrotic disease, and proliferative disease.
  • the present disclosure also provides a method for inhibiting the expression level of HBV genes in cells, the method comprising combining an effective dose of siRNA of the present disclosure, and/or a pharmaceutical composition and/or a siRN conjugate with The cells are in contact.
  • the mechanism of regulating gene expression can be used to prevent and/or treat the for the purpose of a pathological condition or disease.
  • siRNAs, pharmaceutical compositions and/or siRNA conjugates provided by the present disclosure can be used for preventing and/or treating said pathological conditions or diseases, or for the preparation of siRNAs for preventing and/or treating said pathological conditions or diseases described herein.
  • Drug for preventing and/or treating said pathological conditions or diseases, or for the preparation of siRNAs for preventing and/or treating said pathological conditions or diseases described herein.
  • the term "administration/administration” refers to the administration of siRNA, drug composition and/or siRNA conjugate by a method or approach that at least partially localizes siRNA, pharmaceutical composition and/or siRNA conjugate at a desired site to produce a desired effect.
  • the composition and/or siRNA conjugate is placed into a subject.
  • Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, topical administration results in delivery of more of the siRNA, pharmaceutical composition and/or siRNA conjugate to a specific site as compared to the subject's entire body; whereas systemic administration results in delivery of the siRNA, pharmaceutical composition And/or the siRNA conjugate is delivered to substantially the entire body of the subject.
  • a mode of administration capable of delivering the drug to the liver.
  • Administration to a subject may be by any suitable route known in the art, including, but not limited to: oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal Drugs, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual).
  • oral or parenteral routes such as intravenous, intramuscular, subcutaneous, transdermal Drugs, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual).
  • the frequency of administration can be one or more times per day, every week, every two weeks, every three weeks, every month or every year.
  • the dosage of the siRNA, the pharmaceutical composition and/or the siRNA conjugate described in the present disclosure can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, body weight and sex of the subject .
  • Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining the LD50 (the dose that causes 50% of the population to die) and the ED50 (the dose that can cause 50% of the maximum response intensity in quantitative response, and in quantitative response). Middle refers to the dose that causes 50% of the test subjects to have a positive reaction).
  • a range of dosage for use in humans can be derived based on the data obtained from cell culture assays and animal studies.
  • siRNAs When administering siRNAs, pharmaceutical compositions and/or siRNA conjugates described in the present disclosure, for example, for C57BL/6J or C3H/HeNCrlVr mice, male or female, 6-12 weeks old, weighing 18-25g, with The amount of siRNA in the siRNA, pharmaceutical composition and/or siRNA conjugate: for the siRNA conjugate formed by siRNA and a pharmaceutically acceptable conjugating group, the amount of siRNA can be 0.001-100mg/kg Body weight, in some embodiments 0.01-50 mg/kg body weight, in further embodiments 0.05-20 mg/kg body weight, in still further embodiments 0.1-15 mg/kg body weight, in still further embodiments Medium is 0.1-10mg/kg body weight.
  • the above dosages may be preferred.
  • the cells are hepatocytes.
  • the hepatocytes may be cells selected from hepatoma cell lines such as Hep3B, HepG2, Huh7, or isolated primary hepatocytes, in some embodiments primary hepatocytes.
  • the amount of siRNA in the provided siRNA, pharmaceutical composition and/or siRNA conjugate can be easily determined by those skilled in the art according to the desired effect.
  • the amount of siRNA in the provided siRNA conjugates 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 Extracellular concentrations of 0.05 nM to 50 nM or to about 5 nM.
  • the amount necessary to achieve this local concentration will vary depending on various factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, whether the delivery is local or systemic, and the like.
  • the concentration at the site of delivery can be significantly higher than the concentration at the surface of the target cell or tissue.
  • the present disclosure provides a kit comprising the siRNA provided in the present disclosure, and/or the pharmaceutical composition and/or the siRNA conjugate.
  • kits described herein can provide the siRNA, the pharmaceutical composition and/or the conjugate in one container.
  • a kit described herein may comprise a container providing a pharmaceutically acceptable excipient.
  • the kit may also contain other components, such as stabilizers or preservatives.
  • the kits described herein can comprise at least one additional therapeutic agent in a container other than the container providing the siRNA, pharmaceutical composition and/or conjugate described herein.
  • the kit may comprise instructions for mixing the siRNA, pharmaceutical composition and/or conjugate with a pharmaceutically acceptable carrier and/or excipient or other ingredients, if any .
  • the siRNA and the pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate, and/or the pharmaceutically acceptable adjuvant can be provided in any form, for example Liquid form, dry form or lyophilized form.
  • the siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable adjuvant are substantially pure and/or sterile .
  • sterile water can be provided in kits of the present disclosure.
  • reagents and medium used in the following examples are commercially available, and the operations such as nucleic acid electrophoresis and real-time PCR used are all referred to in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)). method to proceed.
  • conjugates 1-16 in the following Table 2 were prepared, the only difference being that the sense strand and antisense strand of siRNA contained in each siRNA conjugate were as shown in Table 2 shown in . According to the nucleic acid sequences of the siRNAs numbered as conjugate 1-conjugate 16 in Table 2 below, the sense strand and the antisense strand of the siRNA were synthesized, respectively.
  • the uppercase letters C, G, U, A, and T represent the base composition of nucleotides;
  • the lowercase letter m indicates that the adjacent nucleotide on the left side 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 nucleotide;
  • the underlined letter combination moe indicates that the nucleotide adjacent to the left of the letter combination moe is ribose 2'-O -Methoxyethyl-modified nucleotides;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by phosphorothioate subunits;
  • the lowercase letter d indicates a nucleus adjacent to the right side of the letter d
  • the nucleotide is a 2'-deoxynucleotide;
  • VP means that
  • each reference siRNA conjugate contains The sense and antisense strands of the siRNAs are shown in Table 2, respectively. According to the nucleic acid sequences of the siRNAs numbered as reference conjugates 1-9 and reference conjugate NC in Table 2 below, the sense and antisense strands of the siRNA were synthesized, respectively.
  • the inventors of the present disclosure measured their double-strand thermal dissociation temperature Tm using the following method.
  • the test method is as follows:
  • the above-prepared conjugate 11 and reference conjugate 9 were prepared as 0.02 mg/mL solutions with 1 ⁇ PBS buffer as the test solution. Add the test solution into a 10mm path length quartz cuvette equipped with a thermal program on the Agilent cary300 UV spectrophotometer, and monitor the temperature-absorbance curve at a wavelength of 260nm, wherein the heating rate is 0.5°C/min, automatically Start at 20.0°C and heat up to 95°C.
  • the double-strand thermal dissociation temperature Tm was calculated from the first derivative of the temperature-absorbance curve according to the specification of the spectrophotometer. Tm value and ⁇ Tm value result are shown in following table 3:
  • ⁇ Tm value (conjugate to be tested) Tm (conjugate to be tested) - Tm (reference conjugate 9);
  • the double-stranded oligonucleotide comprising a stabilized modified nucleotide and the conjugate thereof of the present disclosure have a higher double-stranded oligonucleotide.
  • Chain thermal dissociation temperature compared with the case where the same position is an unmodified nucleotide, the double-stranded oligonucleotide comprising a stabilized modified nucleotide and the conjugate thereof of the present disclosure have a higher double-stranded oligonucleotide.
  • the in vitro psi-CHECK system was used to detect the effect of conjugate 1, conjugate 2, conjugate 3, conjugate 4 and reference conjugate 1 on the target sequence in the in vitro psi-CHECK system. inhibitory activity.
  • modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.
  • Nucle ic Acids Research, 2008.36 (7 ) the method described in 2136-2151 to 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:
  • a detection plasmid was constructed using the psiCHECK TM -2 (Promega TM ) plasmid, which contained a target sequence 1, ie, the siRNA conjugate target sequence.
  • the target sequence 1 is as follows:
  • the target sequence 1 is the complete complementary sequence of the antisense strand in the detected siRNA conjugates, so the inhibitory effect of each siRNA conjugate on the target sequence 1 can reflect the inhibitory ability of the detected siRNA conjugates to target gene expression .
  • the target sequence 1 and its complementary sequence were cloned into the Xho I/Not I site of the psiCHECK TM -2 plasmid.
  • HEK293A cells were seeded in 96-well plates at 8 ⁇ 10 3 cells/well. After 16 hours, when the cell growth density reached 70%, 100 ⁇ L of complete medium in the culture wells was aspirated, and 80 ⁇ L of opti-MEM medium (GIBCO company) to continue culturing for 1.5 hours.
  • opti-MEM medium GEBCO company
  • siRNA conjugate Dilute the above detection plasmid into 20 ⁇ M stock solution with PBS; prepare each siRNA conjugate to be tested to 4 ⁇ M, 1 ⁇ M, 0.25 ⁇ M, 0.0625 ⁇ M, 0.015625 ⁇ M, 0.003906 ⁇ M, 0.0009765 ⁇ M, 0.0002441 ⁇ M, 0.00006104 ⁇ M, 0.00001526 ⁇ M and 0.000003815 ⁇ M (based on the amount of siRNA in the siRNA conjugate) were 11 different concentrations of the siRNA conjugate working solution.
  • the siRNA conjugates used were Conjugate 1, Conjugate 2, Conjugate 3, Conjugate 4 and Reference Conjugate 1 prepared above, respectively.
  • each 1A1-1A11 solution contains 1 ⁇ L of the above-mentioned 11 concentrations of siRNA working solution, 0.05 ⁇ L of detection plasmid working solution (including 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 9.95 ⁇ L of Opti-MEM medium.
  • a part of the 1B solution was mixed with a part of the obtained 1A1-1A11 solution of each siRNA conjugate, and incubated at room temperature for 20 min to obtain a transfection complex 1X1-1X11 of each siRNA conjugate.
  • each siRNA conjugate In the culture well, add the transfection complex 1X1-1X11 of each siRNA conjugate, mix evenly, and add 20 ⁇ L/well to obtain the final concentration of each siRNA conjugate about 40nM, 10nM, 2.5nM respectively , 0.625nM, 0.15625nM, 0.03906nM, 0.009765nM, 0.002441nM, 0.0006103nM, 0.0001526nM, 0.00003815nM (based on the amount of siRNA in the siRNA conjugate) of the transfection complex, the transfection complex of each siRNA conjugate
  • the transfection complexes 1X1-1X11 were all transfected into 3 culture wells to obtain a co-transfection mixture containing siRNA conjugates, which was recorded as the test group.
  • a transfection complex 1 ⁇ 12 was added to the other three culture wells, and the addition amount was 20 ⁇ L/well to obtain a transfection mixture without siRNA conjugate, which was recorded as the blank control group.
  • the co-transfection mixture containing siRNA conjugates and the co-transfection mixture without siRNA conjugates were transfected in culture wells for 4 hours, and each well was supplemented with 100 ⁇ L H-DMEM complete medium containing 20% FBS .
  • the 96-well plate was placed in a CO 2 incubator to continue culturing for 24 hours.
  • the function is as follows,
  • Y is the ratio R, the relative residual activity of Renilla
  • X is the logarithmic value of transfection siRNA concentration
  • Bot is the Y value at the bottom of the steady state period
  • Top is the Y value at the top of the steady state period
  • X' is the X value corresponding to when Y is halfway between the bottom and the top
  • HillSlope is the slope of the curve at X'.
  • conjugates 1-4 and reference conjugate 1 is: according to the direction from the 5' end to the 3' end, the base on the 18th nucleotide of the sense strand of conjugate 1 is C,
  • the third nucleotide of the antisense strand has a 2'-O-methoxyethyl modification group; the base on the 18th nucleotide of the sense strand of conjugate 2 is G, and the base of the antisense strand There is a 2'-O-methoxyethyl modification group on the third nucleotide; the base on the 18th nucleotide of the sense strand of conjugate 3 is C, and the fifth core of the antisense strand There is a 2'-O-methoxyethyl modification group on the nucleotide; the base on the 18th nucleotide of the sense strand of conjugate 4 is G, and the base on the fifth nucleotide of the antisense strand has 2'-O-methoxye
  • the siRNA conjugates of the present disclosure have high target sequence inhibitory activity in the psi-CHECK system in vitro, with IC 50 between 0.01-0.05nM.
  • the siRNA conjugates of the present disclosure have comparable target sequence inhibitory activity compared with reference conjugate 1 that does not contain stabilizing modified nucleotides, and even have higher target sequence inhibitory activity than reference conjugate 1. active.
  • Target sequence 2 5'-GCAGCTTCTTAGGTAGGCATATTGGGCAGCTTCTTAGGTAGGCATATTGGGCAGCTTCTTAGGTAGGCATATTGGGCAGCTTCTTAGGTAGGCATA-3' (SEQ ID NO: 234);
  • the target sequence 2 contains a sequence complementary to the antisense strand of the siRNA in the conjugate to be tested, so the inhibitory effect of the conjugate to be tested on the target sequence 2 can reflect the degree of off-target effects. That is, the higher the inhibitory effect, the more likely the conjugate to be tested is off-target.
  • the measured IC50 values are summarized in Table 5.
  • IC 50 /(MOS-5) /IC 50 /(GSCM) can reflect the anti-off-target effect that different siRNA conjugates can achieve when they achieve the same target sequence inhibitory activity.
  • siRNA conjugates of the present disclosure all showed greater IC 50 /(MOS-5) /IC 50 / (GSCM) values, especially conjugate 2, IC50 /(MOS-5) / IC50/(GSCM) value is 3.4 times that of reference conjugate 1.
  • siRNA Conjugates 1-4 of the present disclosure had significantly reduced off-target effects while achieving the same target sequence inhibitory activity.
  • Target sequence 3 5'-GGCTTTCAGCTATATGGAT-3' (SEQ ID NO: 235).
  • Conjugate 5 Conjugate 7 and Reference Conjugate 2
  • the base on the 18th nucleotide of the sense strand of Conjugate 5 according to the direction from the 5' end to the 3' end C, the third nucleotide of the antisense strand has a 2'-O-methoxyethyl modification group
  • the base on the 18th nucleotide of the sense strand of conjugate 7 is C
  • the base of the antisense strand is C
  • the base on the 18th nucleotide of the sense strand of reference conjugate 2 is A
  • the base on the antisense strand There is no 2'-O-methoxyethyl modification group on it.
  • the siRNA conjugates of the present disclosure have high target sequence inhibitory activity in the psi-CHECK system in vitro, with IC 50 between 0.01-0.1 nM.
  • the siRNA conjugates of the present disclosure have comparable target sequence inhibitory activity compared with the reference conjugate 2 that does not contain stabilizing modified nucleotides, and the conjugate 7 even has a higher activity than the reference conjugate 2. High target sequence inhibitory activity.
  • Target sequence 4 5'-GGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTT-3' (SEQ ID NO: 236);
  • the target sequence 4 contains a sequence complementary to the antisense strand of the siRNA in the conjugate to be tested, so the inhibitory effect of the conjugate to be tested on the target sequence 4 can reflect the degree of off-target effect. That is, the higher the inhibitory effect, the more likely the conjugate to be tested is off-target.
  • the measured IC50 values are summarized in Table 7.
  • mice Primary mouse liver cells were isolated from C57BL/6j-TgN (AlblHBV) 44Bri mice (referred to as 44Bri mice for short). Add 1 ⁇ 105 cells/well mouse liver primary cell solution to each well of the 12-well culture plate, and culture the cell plate overnight in a 37°C, 5% CO 2 incubator.
  • a conjugate stock solution with a concentration of 20 ⁇ M (based on the amount of siRNA in the conjugate) was prepared with 1 ⁇ PBS. Then it was diluted to obtain a working solution of the conjugate with a concentration of 4 ⁇ M.
  • each test conjugate was added to 2 culture wells in parallel.
  • RNA in cells in each well was extracted according to the method described in the manual.
  • RNA For each well of cells, take 1 ⁇ g of total RNA, and use the reagents provided by the reverse transcription kit Goldenstar TM RT6 cDNA Synthesis Kit (purchased from Promega, Cat. The total RNA of cells in each well was reverse-transcribed in the System kit instructions. After the reaction, 80 ⁇ L of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.
  • each reverse transcription reaction system For each reverse transcription reaction system, take 5 ⁇ L of the above cDNA-containing solution as a template, use SYBR qPCR SuperMix Plus kit (purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B) provides 20 ⁇ L of reagents to configure the qPCR reaction system.
  • the PCR primer sequences used to amplify the target gene HBV and the internal reference gene GAPDH are shown in Table 8 As indicated, the final concentration of each primer was 10 ⁇ M.
  • Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s. After repeating the above-mentioned denaturation, annealing and extension process 40 times in total, the product W1 containing amplified target gene HBV and internal reference gene GAPDH was obtained.
  • the product W1 then went through 95°C for 15s, 60°C for 1min, gradient heating to 95°C and collecting fluorescence signal every 0.3°C, 95°C for 15s, real-time fluorescence quantitative PCR instrument respectively collected the melting curves of the target gene and the internal reference gene GAPDH in the product W1 , to obtain the Ct values of the target gene HBV and the internal reference gene GAPDH.
  • Adopt comparative Ct ( ⁇ Ct) method carry out relative quantitative calculation to the expression level of target gene HBV mRNA in each test group, calculation method is as follows:
  • ⁇ Ct (test group) Ct (target gene of test group) – Ct (internal reference gene of test group)
  • ⁇ Ct (control group) Ct (control group target gene) – Ct (control group internal reference gene)
  • ⁇ Ct (test group) ⁇ Ct (test group) - ⁇ Ct (average of the control group)
  • ⁇ Ct (control group) ⁇ Ct (control group) - ⁇ Ct (control group average)
  • ⁇ Ct average of control group
  • ⁇ Ct control group
  • the expression level of HBV mRNA in the test group is normalized, and the mean value of the HBV mRNA expression level in the control group is defined as 100%.
  • Test group HBV mRNA relative expression level 2 - ⁇ Ct (test group) ⁇ 100%
  • Test group HBV mRNA suppression rate (1-test group HBV mRNA relative expression level) ⁇ 100%
  • FIG. 1 is a bar graph showing the target sequence inhibitory activity of the conjugate of the present disclosure and the reference conjugate in mouse liver primary cells at a concentration of 10 nM.
  • the results show that when the conjugates 1-4, conjugate 5 and conjugate 7 of the present disclosure have a better inhibitory activity in vitro at a concentration of 10 nM, especially conjugate 2 and conjugate 4, the inhibitory activity Up to 99.4%.
  • the conjugate containing the stabilizing modified nucleotide showed substantially the same or even higher activity.
  • Hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunoassay) (Shanghai Kehua Biology) was used to detect the serum HbsAg content of 44Bri mice according to the method recorded in the instructions, and the mice with S/COV>10 were selected and randomly grouped (both Male), 5 mice in each group, numbered respectively, administered the conjugate 10 or the reference conjugate 3 to each mouse at a dose of 1 mg/kg mouse body weight (calculated as siRNA) by subcutaneous injection , the siRNA conjugate was provided in the form of a 1 ⁇ PBS solution containing 0.2 mg/ml (calculated as siRNA) of the siRNA conjugate, and the administration volume was 5 ml/kg; each of the other two groups of mice was given 1 ⁇ PBS with an administration volume of 5ml/kg was used as a blank control group.
  • siRNA mouse body weight
  • RNA later (Sigma Aldrich); 1mL Trizol (Sigma Company) was added to each liver tissue. ), crushed 3 times in a Tissuelyset II automatic tissue homogenizer, each time for 30s, to obtain liver tissue homogenate, add 0.2mL chloroform to it, and let it stand for 3min. Centrifuge at 12,000 rpm for 10 min at 4°C, and take 0.4 mL of the supernatant. Add 0.5mL isopropanol to the supernatant and let it stand at room temperature for 10min.
  • RNA in the liver tissue of each mouse take 10.5 ⁇ L of total RNA aqueous solution containing 1 ⁇ g of total RNA, use the reverse transcription kit Reverse Transcription System (purchased from Promega, Cat. No. A3500), and reverse The recording operation steps were prepared as 20 ⁇ L of reverse transcription reaction system, and the total RNA was reverse transcribed.
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 42°C for 30 minutes, then incubate at 95°C for 5 minutes, and finally incubate at 4°C for 5 minutes. Add 80 ⁇ L of DEPC water to the recording reaction system to obtain a solution containing cDNA.
  • each reverse transcription reaction system takes 5 ⁇ L of the above cDNA-containing solution as a template, and use the reagents provided by the SYBR select Master Mix kit (Applied biosystem company) to prepare 20 ⁇ L of a qPCR reaction system, which is used to amplify the target gene HBV
  • the PCR primer sequences of X and the internal reference gene GAPDH are shown in Table 9 below, and the final concentration of each primer is 0.25 ⁇ M.
  • Each qPCR reaction system was placed on the ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s. After repeating the above-mentioned processes of denaturation, annealing and extension a total of 40 times, a product W containing amplified target gene HBV X and internal reference gene GAPDH was obtained. The product W was then incubated at 95°C for 1min, 55°C for 30s, and 95°C for 30s. The real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene HBV X and the internal reference gene GAPDH in the product W respectively, and obtained the target gene HBV X and the internal reference gene GAPDH. Ct value.
  • Adopt comparative Ct ( ⁇ Ct) method carry out relative quantitative calculation to the expression level of target gene HBV mRNA in each test group, calculation method is as described in above-mentioned experimental example 5.
  • Figure 2 is a scatter diagram of the relative expression level of HBV mRNA in the liver of 44Bri mice given 1 mg/kg (calculated as siRNA) of conjugate 10 or reference conjugate 3 and PBS.
  • PBS in the figure represents the blank control group.
  • the inhibition rate of each siRNA conjugate to HBV mRNA is summarized in Table 10.
  • conjugate 10 has a 2'-O-formazan Oxyethyl modification group; there is no 2'-O-methoxyethyl modification group on the antisense strand of reference conjugate 3.
  • the siRNA conjugates of the present disclosure show excellent HBV mRNA inhibitory effect in mice, and at a dose of 1 mg/kg, the HBV mRNA inhibitory rate of conjugate 10 in mice can be Up to 84.25%, showing an HBV mRNA inhibitory activity similar to that of the reference conjugate 3 that does not include stabilizing modified nucleotides.
  • Conjugate 9, Conjugate 10, Reference Conjugate 3 and Reference Conjugate 4 were dissolved in PBS to a 3 mg/ml solution (calculated as siRNA conjugate).
  • the ICR mice half male and half male, weighing 18-22 g, 5-6 weeks old, purchased from Speyford Co.
  • mice were randomly divided into groups, with 6 mice in each group (half male and half male), and numbered respectively.
  • the above-mentioned siRNA conjugate solution was administered to each mouse, and the administration volume was 10 mL/kg, as a test group; in addition, PBS was administered to each of a group of mice, and The drug volume was 10mL/kg, which was used as a blank control group.
  • the orbital blood was collected from each mouse in the test group and the blank control group.
  • the blood collection volume was 0.6 mL. Centrifuge at 3000rpm for 15min to obtain serum.
  • concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum was further detected by PM1P000/3 automatic serum biochemical analyzer (SABA, Italy). See Figure 3A and Figure 3B for the results.
  • PBS represents the blank control group.
  • Figure 3A and Figure 3B are the scatter points of ALT and AST concentrations in mouse serum after administration of 30 mg/kg of conjugate 9, conjugate 10, reference conjugate 3, reference conjugate 4 or PBS, respectively picture. It can be seen from Figure 3A and Figure 3B that, compared with the blank control group, after administration of the reference conjugate 3 that does not contain stabilizing modified nucleotides, serum ALT and AST concentrations increased; while administration of the siRNA conjugate of the present disclosure After the siRNA conjugate, the serum ALT and AST concentrations were comparable to those of the blank control group, indicating that the siRNA conjugate of the present disclosure has low liver toxicity.
  • the concentrations of ALT and AST in mouse serum were significantly increased, indicating that the The reference conjugate may produce higher hepatotoxicity.
  • mice were sacrificed and dissected, preserved in 10% neutral buffered formalin fixative, and pathological sections were made. The severity of hepatic steatosis in the pathological sections was evaluated and graded, and a relative comparison was made.
  • mice administered with the reference conjugate 3 that did not contain stabilized modified nucleotides 4 mice exhibited severe hepatocyte degeneration, specifically manifested by extensive liver tissue.
  • the cytoplasm of the cells was loose, and many hepatocytes were balloon-like degeneration, the cells were swollen, and the cytoplasm was vacuolated.
  • One mouse showed moderate hepatocyte degeneration. There was vacuolar degeneration, tiny round vacuoles were seen in the cytoplasm, a small number of local hepatocytes were necrotic, and the nuclei were condensed or fragmented.
  • One mouse showed mild hepatocyte degeneration, specifically manifested as more hepatocyte Hepatic steatosis was more severe than that in the blank control group.
  • mice given the reference conjugate 4 containing a stabilizing modified nucleotide only at the 7th position of the antisense strand 5'-3' end direction showed severe hepatic steatosis,
  • Four showed moderate hepatic steatosis and also showed more severe hepatic steatosis than in the mice of the blank control group.
  • mice administered with siRNA conjugate 9 of the present disclosure 2 mice showed moderate hepatic steatosis, 3 mice showed mild hepatic steatosis, and no severe or severe hepatocyte degeneration was observed.
  • mice administered with siRNA conjugate 10 of the present disclosure 3 mice showed moderate hepatic steatosis, 2 mice showed mild hepatic steatosis, and no severe or severe hepatocyte degeneration was seen.
  • Mice administered the conjugates of the present disclosure exhibited a lower degree of hepatic steatosis compared to the reference conjugate.
  • the siRNA conjugates of the present disclosure can effectively reduce the liver toxicity caused by off-target effects, so in the preparation of drugs for the treatment and/or prevention of HBV diseases or symptoms Shows significantly higher safety and has excellent development prospects.
  • HBV transgenic mice 44Bri/J were purchased from the Department of Experimental Animal Science, Peking University Health Science Center, and mice with S/COV>10 were selected for the experiment before the experiment.
  • Primary mouse liver cells were extracted from fresh liver tissue of 44Bri mice, and the density of primary mouse liver cells was adjusted to 1 ⁇ 105 cells in Opti-MEM (1X) medium (GIBCO, Cat. No. 31985-070) /mL to obtain mouse liver primary cell suspension. Then, the obtained mouse liver primary cell suspensions were respectively added to different culture wells of the 12-well plate, and the mouse liver primary cells were inoculated into the culture wells. The volume of the primary mouse liver cell suspension added was 1 mL/well, and the number of primary mouse liver cells was 1 ⁇ 10 5 cells/well.
  • Opti-MEM (1X) medium GIBCO, Cat. No. 31985-070
  • siRNA conjugate working solutions 4 ⁇ M (calculated as siRNA) siRNA conjugate working solutions, and the siRNA conjugates used are Conjugate 11, Conjugate 12 or Reference than conjugate 5.
  • the reference conjugate NC was formulated into a 4 ⁇ M reference siRNA NC working solution.
  • siRNA conjugate working solution or the reference conjugate NC working solution of each conjugate to the above-mentioned culture wells of primary mouse liver cell suspension, mix evenly, and add 2.5 ⁇ L/well , each siRNA conjugate or reference conjugate NC were transfected into 3 culture wells to obtain a transfection mixture containing siRNA (calculated as siRNA, with a final concentration of 10 nM), which was recorded as the test group.
  • the mouse liver primary cell suspension in the other 3 culture wells was recorded as the blank control group.
  • Each transfection mixture containing siRNA and the blank control group were placed in an incubator containing 5% CO 2 and incubated at 37° C. for 24 h.
  • TRIZOL purchased from SIGMA, product number T9424 was used to extract the total RNA in the cells in each well according to the method described in the manual to obtain an aqueous solution of total RNA.
  • RNA aqueous solution containing 1 ⁇ g of total RNA was taken respectively, and the reagents provided by the reverse transcription kit Goldenstar TM RT6 cDNA Synthesis Kit (purchased from Beijing Qingke Xinye Biotechnology Co., Ltd., catalog number TSK301M) were used.
  • Goldenstar TM Oligo(dT) 17 was used as a primer, and 20 ⁇ L of a reverse transcription reaction system was configured according to the reverse transcription operation steps in the kit manual, and the total RNA of cells in each well was reverse transcribed.
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 50°C for 50 minutes, then incubate at 85°C for 5 minutes, and finally incubate at 4°C for 5 minutes. Add 80 ⁇ L of DEPC water to the system to obtain a solution containing cDNA.
  • each reverse transcription reaction system takes 5 ⁇ L of the above cDNA-containing solution as a template, use The reagents provided by the SYBR qPCR SuperMix Plus kit (purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B) prepared 15 ⁇ L of the qPCR reaction system, in which the PCR primer sequences used to amplify the target gene HBV X and the internal reference gene GAPDH are shown in the table 9, the final concentration of each primer was 0.25 ⁇ M.
  • SYBR qPCR SuperMix Plus kit purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B
  • the final concentration of each primer was 0.25 ⁇ M.
  • Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 25s, and extension at 72°C for 25s. After repeating the above-mentioned denaturation, annealing, and extension processes for a total of 40 times, a product W containing amplified target gene HBV X and internal reference gene GAPDH was obtained. The product W was then incubated at 95°C for 1min, 55°C for 30s, and 95°C for 30s. The real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene HBV X and the internal reference gene GAPDH in the product W respectively, and obtained the target gene HBV X and the internal reference gene GAPDH. Ct value.
  • Figure 4 is a histogram of the relative expression levels of HBV mRNA in primary hepatocytes of 44Bri mice after freely ingesting Conjugate 11, Conjugate 12 or Reference Conjugate 5 and Reference Conjugate NC respectively. Further, the inhibition rate of each siRNA conjugate or reference conjugate NC to HBV mRNA is summarized in Table 11.
  • conjugate 11, conjugate 12 and reference conjugate 5 The difference between conjugate 11, conjugate 12 and reference conjugate 5 is that according to the direction from the 5' end to the 3' end, the third nucleotide of the antisense strand of conjugate 11 has 2 '-O-methoxyethyl modification group; the fifth nucleotide of the antisense strand of conjugate 12 has a 2'-O-methoxyethyl modification group; reference conjugate 5 antisense There is no 2'-O-methoxyethyl modification group on the sense strand.
  • the siRNA conjugates of the present disclosure exhibit excellent HBV mRNA inhibitory activity in 44Bri mouse primary liver cells, and at an siRNA concentration of 10 nM, the HBV mRNA inhibitory rate is at least 91.77%;
  • the HBV mRNA inhibition rate of conjugate 11 in primary mouse hepatocytes was up to 93.06%, showing substantially equivalent HBV mRNA inhibition to the corresponding reference conjugate 5 that did not include the stabilizing modified nucleotide active.
  • Hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunoassay) (Shanghai Kehua Biology) was used to detect the serum HbsAg content of 44Bri mice according to the method recorded in the instructions, and the mice with S/COV>10 were selected and randomly grouped (both male), 5 mice in each group, numbered respectively, administered the conjugate 11, conjugate 2 or reference to each mouse at a dose of 1 mg/kg mouse body weight (siRNA) by subcutaneous injection Compared with conjugate 5, the siRNA conjugate was provided in the form of 1 ⁇ PBS solution containing 0.2mg/ml (calculated as siRNA) of the siRNA conjugate, and the administration volume was 5ml/kg; Each of them was given 1 ⁇ PBS, and the administration volume was 5ml/kg, which served as a blank control group.
  • siRNA mouse body weight
  • RNA later (Sigma Aldrich); 1mL Trizol (Sigma Company) was added to each liver tissue. ), crushed 3 times in a Tissuelyset II automatic tissue homogenizer, each time for 30s, to obtain liver tissue homogenate, add 0.2mL chloroform to it, and let it stand for 3min. Centrifuge at 12,000 rpm for 10 min at 4°C, and take 0.4 mL of the supernatant. Add 0.5mL isopropanol to the supernatant and let it stand at room temperature for 10min.
  • RNA in the liver tissue of each mouse take 10.5 ⁇ L of total RNA aqueous solution containing 1 ⁇ g of total RNA, use the reverse transcription kit Reverse Transcription System (purchased from Promega, Cat. No. A3500), and reverse The recording operation steps were prepared as 20 ⁇ L of reverse transcription reaction system, and the total RNA was reverse transcribed.
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 42°C for 30 minutes, then incubate at 95°C for 5 minutes, and finally incubate at 4°C for 5 minutes. Add 80 ⁇ L of DEPC water to the recording reaction system to obtain a solution containing cDNA.
  • each reverse transcription reaction system takes 5 ⁇ L of the above cDNA-containing solution as a template, and use the reagents provided by the SYBR select Master Mix kit (Applied biosystem company) to prepare 20 ⁇ L of a qPCR reaction system, which is used to amplify the target gene HBV
  • the PCR primer sequences of X and the internal reference gene GAPDH are shown in Table 9, and the final concentration of each primer is 0.25 ⁇ M.
  • Each qPCR reaction system was placed on the ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s. After repeating the above-mentioned processes of denaturation, annealing and extension a total of 40 times, a product W containing amplified target gene HBV X and internal reference gene GAPDH was obtained. The product W was then incubated at 95°C for 1min, 55°C for 30s, and 95°C for 30s. The real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene HBV X and the internal reference gene GAPDH in the product W respectively, and obtained the target gene HBV X and the internal reference gene GAPDH. Ct value.
  • Figure 5 is a scatter diagram of the relative expression level of HBV mRNA in the liver of 44Bri mice given 1 mg/kg (calculated as siRNA) of Conjugate 11, Conjugate 12 or Reference Conjugate 5 and PBS.
  • PBS represents a blank control group.
  • the inhibition rate of each siRNA conjugate to HBV mRNA is summarized in Table 12.
  • the siRNA conjugates of the present disclosure exhibit excellent HBV mRNA inhibitory effects in mice, and at a dose of 1 mg/kg, the HBV mRNA inhibitory rate can be as high as 96.31%, and it is shown to be as high as the corresponding Comparable HBV mRNA inhibitory activity of reference conjugate 5 excluding stabilizing modified nucleotides.
  • Conjugate 11 Conjugate 12, Reference Conjugate 5 and Reference Conjugate 8 were dissolved in PBS to a 6 mg/ml solution (calculated as siRNA conjugate).
  • SD rats all male, 0.22-0.28 kg in weight, 5-7 weeks old, purchased from Victoria Lihua Company
  • the above-mentioned siRNA conjugate solution was administered to each rat respectively, and the administration volume was 5mL/kg, as a test group; in addition, PBS was administered to each of a group of rats, and Drug volume is 5mL/kg, as a blank control group.
  • each rat in the test group and the blank control group was killed on the 15th day and dissected, and the liver was weighed and normalized based on the blank control group, at 10% Preserve in neutral buffered formalin fixative and make pathological sections.
  • the results of gross autopsy and liver weight are shown in Table 13.
  • the severity of hepatic steatosis and inflammation were evaluated and graded and compared.
  • % and the preceding numbers represent the percentage difference between the corresponding index and the reference blank control group.
  • ⁇ stands for increase.
  • ⁇ 5.43% means that the liver weight increased by 5.43% compared with the blank control group.
  • Conjugate 11 Conjugate 12, Reference Conjugate 5 and Reference Conjugate 8
  • the third antisense strand of Conjugate 11 There is a 2'-O-methoxyethyl modification group on the first nucleotide; there is a 2'-O-methoxyethyl modification group on the fifth nucleotide of the antisense strand of conjugate 12; There is no 2'-O-methoxyethyl modification group on the antisense strand of reference conjugate 5; there are 2'-O-methoxyethyl modification groups on the seventh and twelfth nucleotides of the antisense strand of reference conjugate 8 '-O-methoxyethyl modification group.
  • All rats administered with the reference conjugate 8 showed extremely severe hepatic steatosis and mild or moderate liver inflammation, specifically manifested as extensive and severe fatty degeneration of hepatocytes in the tissue, and varying amounts, Round vacuoles of different sizes, several small focal infiltrations of inflammatory cells in the lobules or occasional infiltration of inflammatory cells around the portal vein, severe swelling of a small number of hepatocytes, and light cytoplasmic staining.
  • the siRNA conjugates of the present disclosure can effectively reduce the liver toxicity caused by off-target effects, so in the preparation of drugs for the treatment and/or prevention of HBV diseases or symptoms Shows significantly higher safety and has excellent development prospects.
  • Multiple segments of the target sequence 5 are partially complementary to the antisense strand of the siRNA in the siRNA conjugate to be tested, so the inhibitory effect of each siRNA conjugate on the target sequence 3 can reflect the degree of off-target effect. That is, the higher the inhibitory effect, the more likely the siRNA conjugate is off-target.
  • the off-target IC50 value of reference conjugate 5 was 218.085pM
  • the off-target IC50 value of reference conjugate 9 was 202.581pM, that is, the two reference siRNA conjugates were above the off-target IC50
  • the relative residual activity of Renilla was always higher than 50%, among which, the relative residual activities of Conjugate 11, Conjugate 12, Conjugate 13, Conjugate 14 and Conjugate 16 were the lowest in order of 55.65%, 71.42%, 71.38%, 65.47%, 67.84%, that is, none of the above siRNA conjugates occurred off-target. It can be seen that, compared with reference conjugate 5 and reference conjugate 9 that do not contain stabilizing modified nucleotides, each siRNA conjugate
  • Figure 6 shows that conjugate 11, conjugate 12, conjugate 15, conjugate 16, reference conjugate 5, reference conjugate 9, reference conjugate 6, reference Histogram of relative expression levels of HBV mRNA in primary hepatocytes of 44Bri mice after conjugate 7 or reference conjugate NC. Further, the inhibition rate of each siRNA conjugate or reference conjugate NC to HBV mRNA is summarized in Table 14.
  • siRNA conjugates 11, 12, 15 and 16 of the present disclosure showed excellent HBV mRNA inhibitory activity in 44Bri mouse primary liver cells, and at a siRNA concentration of 10 nM, HBV The mRNA inhibition rate is at least 78.47%, and the highest can reach 85.97%, and the HBV mRNA inhibition activity is comparable to that of the reference conjugate 5, and significantly higher than that of the reference conjugates 9, 6 and 7.
  • the corresponding position in reference conjugate 5 is a non-stabilizing modified nucleotide
  • the corresponding position in reference conjugate 9 is an unmodified nucleotide
  • reference conjugates 6 and 7 are on the antisense strand In addition to the 3-9 position in the direction of the 5'-3' end, it also contains stabilizing modified nucleotides.

Abstract

一种能够抑制乙型肝炎病毒(HBV)基因表达的 siRNA，包含正义链和反义链，所述正义链和反义链各自独立地包含由 19 个修饰或未修饰的核苷酸组成的核苷酸序列Ⅰ或核苷酸序列I，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HBV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，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。提供的 siRNA以及包含该siRNA的药物组合物和siRNA缀合物可以治疗和/或预防与HBV 基因表达相关的疾病或症状，并降低脱靶效应。

Description

一种核酸、含有该核酸的组合物与缀合物及其用途 技术领域
本公开涉及一种siRNA、含有该siRNA的药物组合物和缀合物及其用途。具体地，本公开涉及一种用于抑制乙型肝炎病毒(hepatitis B virus,HBV)基因表达siRNA、含有该siRNA作为活性成分的药物组合物和缀合物，以及该siRNA、药物组合物和siRNA缀合物在制备用于治疗和/或预防乙型肝炎的药物中的用途。
背景技术
乙型病毒性肝炎(又称乙型肝炎或乙肝)是严重威胁全球、特别是中国的一类传染病。目前全球公认的两大类乙肝防治药物为干扰素和核苷类似物，但是这两类药物存在使用后容易产生耐药性或使用受限等多种弊端。例如，干扰素容易产生不良反应、核苷类药物存在耐药性和停药后复发的问题。因此，若能从基因水平上沉默病毒的基因表达，阻断HBV的生成和复制，由此从根本上降低病毒代谢和对肝细胞的侵染，无疑将是最为理想的乙肝治疗手段。从理论上讲，小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNAinterference，RNAi)这一机制，以序列特异性的方式抑制或阻断任何感兴趣的目的基因(例如引发如癌症等疾病的基因)的表达，从而达到治疗疾病的目的。
另一方面，在siRNA的成药研究中，脱靶效应是与毒性相关的重要副作用之一。目前，许多在临床前药学研究中显示出优异的药学活性的siRNA由于其脱靶效应产生的毒性而难以用于实际的药物研发。而理想的、期望用于制备试剂应用于患者的药物siRNA无疑应具有低毒性，包括低的由脱靶效应导致的毒性。因此，在本领域中如何获得具有低脱靶效应的siRNA仍需进一步深入探索。
PCT国际申请WO2016077321A1中公开了众多特异性靶向HBV基因X基因区的siRNA以及对其进行递送的方法，并通过对siRNA的核苷酸进行修饰，从而增强了其在血浆中的稳定性。
鉴于修饰方案对siRNA的重要性，仍然需要寻找新的siRNA修饰方案，以进一步增强siRNA的稳定性，并保持其活性。
发明内容
为了开发一种具有显著低的脱靶效应、能够抑制HBV基因的siRNA，发明人出乎意料地发现，在序列特定位置处具有稳定化修饰核苷酸的siRNA显示出比对应位置不具有稳定化修饰核苷酸的siRNA显著更低的脱靶效应，并 且还显示出未明显降低、相当甚至更高的HBV基因抑制活性。
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HBV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中的每一个核苷酸独立地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HBV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2'-O-甲氧基乙基修饰的核苷酸。
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连接或非共价连接，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，本公开还提供了一种药物组合物，该药物组合物含有本公开提供的siRNA，和/或本公开提供的缀合物以及药学上可接受的载体。
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HBV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HBV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HBV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缀合物具有良好的稳定性和低的脱靶效应，并具有良好的HBV基因表达的抑制活性，具体说明如下。
第一，本公开的siRNA、药物组合物和/或siRNA缀合物可在体外具有更低的脱靶效应和/或由于脱靶效应导致的毒性反应。本公开提供的siRNA缀合物，在siRNA的给药浓度为40nM时，并且将与待测缀合物中siRNA反义链部分互补的序列扩增5倍的情况下，仍检测不到siRNA缀合物的脱靶，本公开的缀合物显示出与不包含稳定化修饰核苷酸的参比缀合物相比更显著的降低脱靶的效应。又例如，以30mg/kg剂量给予本公开的siRNA缀合物的大鼠中，肝重几乎不增加，并且在在肝脂肪变性和炎症方面显示出明显低于参比siRNA缀合物的毒性反应。又例如，本公开提供的siRNA缀合物在体外sicheck系统中对脱靶目标序列抑制率始终不高于25％，与不包含稳定化修饰核苷酸的参比siRNA缀合物相比显示出显著更低的脱靶效应。又例如，以30mg/kg剂量给予本公开的siRNA缀合物的小鼠中，在肝脂肪变性方面显示出与空白对照组相接近的毒性反应，并显著低于给予不包含稳定化修饰核苷酸的参比缀合物的小鼠显示的肝脂肪变性。进一步地，在病理切片中，给予本公开的siRNA缀合物的小鼠中在肝脂肪变性和炎症方面同样显示出与空白对照组相接近的反应，没有显著异常，表明本公开的siRNA缀合物具有很低的肝毒性。又例如，与不包含稳定化修饰核苷酸的参比siRNA缀合物相比，本公开的siRNA缀合物不仅对在靶目标序列的抑制活性相当或显著更高，还均显示出显著更低的脱靶效应。又例如，与给予参比siRNA缀合物的小鼠相比，以30mg/kg剂量给予本公开的siRNA缀合物的小鼠中，血生化指标显著降低，与空白对照组保持接近的水平；并且在组织病理学中显示出显著更低的毒性反应，多数小鼠中未发现异常，而仅有部分小鼠出现轻度的肝细胞变性。
第二，本公开的siRNA、药物组合物和/或siRNA缀合物在体外实验中显示出优异的HBV基因调节活性。例如，本公开提供的的缀合物在10nM浓度时，都具有较好的体外抑制活性，特别是缀合物2和缀合物4，抑制活性高达 99.4％，与不包含稳定化修饰核苷酸的参比缀合物相比，可表现出基本相同甚至更高的活性。又例如，本公开的siRNA缀合物在44Bri小鼠原代肝细胞中显示出优异的HBV mRNA抑制活性，在10nM的siRNA浓度下，HBV mRNA抑制率至少为81.29％，最高可达93.06％，并显示出与对应不包括稳定化修饰核苷酸的参比缀合物5相当的HBV mRNA抑制活性；并且与对应位置是未修饰核苷酸的参比siRNA缀合物相比，HBV mRNA抑制活性出人意料地均有显著增加，最高可增加22.99％。又例如，在体外psi-CHECK系统中，与参比缀合物相比，本公开的缀合物均显示出与不包含稳定化修饰核苷酸的参比缀合物相比具有相当的目标序列抑制活性，例如，本公开的缀合物具有比参比缀合物更小的IC 50/(GSCM)值，IC 50/(GSCM)值降低了0.0137-0.0261nM之间，相较于参比缀合物，缀合物的IC 50/(GSCM)值降低了29.5％-56.3％。并且，本公开的缀合物均显示出与不包含稳定化修饰核苷酸的参比缀合物相比更大的IC 50/(MOS-5)/IC 50/(GSCM)值，尤其是缀合物2，IC 50/(MOS-5)/IC 50/(GSCM)值是参比缀合物1的3.4倍。
由此说明，本公开提供的siRNA、药物组合物以及siRNA缀合物具有显著更低的脱靶效应以及由于脱靶效应引起的毒性反应，还能够在体外有效抑制HBV基因的表达，因此可在具有显著更高的安全性的同时，有效治疗和/或预防由HBV基因表达的mRNA水平相关的疾病或症状，具有良好的应用前景。
附图说明
图1为10nM浓度的本实施例的缀合物、参比缀合物在小鼠肝原代细胞中的目标序列抑制活性的柱状图。
图2为分别给予了本实施例的缀合物或参比缀合物以及PBS后，44Bri小鼠肝内HBV mRNA相对表达水平的散点图。
图3A和图3B为分别给予了本实施例的缀合物或参比缀合物以及PBS后，小鼠血清中ALT和AST浓度的散点图。
图4为分别自由摄取了本公开的siRNA缀合物或参比siRNA缀合物以及参比缀合物NC后，44Bri小鼠肝原代细胞中HBV mRNA相对表达水平的柱状图。
图5为给予了本公开的siRNA缀合物或参比siRNA缀合物以及PBS后，44Bri小鼠肝内HBV mRNA相对表达水平的散点图。
图6为分别自由摄取了本公开的siRNA缀合物或参比siRNA缀合物以及参比缀合物NC后，44Bri小鼠肝原代细胞中HBV mRNA相对表达水平的柱状图。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
在本公开中，如无其他说明，HBV mRNA是指具有如Genbank注册号NC_003977.1所示序列的mRNA，HBV基因是指转录上述HBV 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亚基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸，在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。
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，所述“反向互补”指在双链核酸分子中，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。
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)。
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能度对特定的反应条件不敏感，并且可以在分子中的该官能度上添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2,2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献各自整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基氧杂蒽-9-基(Pixyl)和9-(对甲氧基苯基)氧杂蒽-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4'-二甲氧基三苯甲基)和TMTr(4,4',4”-三甲氧基三苯甲基)。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、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”指的是获得有益的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。
本公开的siRNA
在一方面，本公开提供了一种具有较高的HBV基因抑制活性、且具有低的脱靶效应的siRNA。
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中的每一个核苷酸独立地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HBV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的双链热解离温度(Tm)升高。在一些实施方式中，“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siRNA的Tm升高0.1-6℃。在一些实施方式中指siRNA的Tm升高0.5-4℃。不受理论解释限制地，通过在特定位置包含稳定化修饰核苷酸，本公开的siRNA中反义链与HBV基因表达的mRNA结合能力基本不受影响，而与脱靶目标mRNA之间的结合显著降低，从而降低甚至消除脱靶效应。
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为C 2-C 6烷基、取代的C 2-C 6烷基、C 6-C 8芳基、取代的C 6-C 8芳基中的一种，每个R'独立地为H、C 1-C 6烷基、取代的C 1-C 6烷基、C 6-C 8芳基、取代的C 6-C 8芳基中的一种，所述取代的C 2-C 6烷基、取代的C 6-C 8芳基或取代的C 1-C 6烷基是指C 2-C 6烷基、C 6-C 8芳基或C 1-C 6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：C 1-C 3烷基、C 6-C 8芳基、C 1-C 3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， 而仅涉及那些能够实现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为2'-O-甲氧基乙基。
在另一方面，本公开还提供了一种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中的每一个核苷酸独立地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HBV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2'-O-甲氧基乙基修饰的核苷酸。
在一些实施方式中，本公开的siRNA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2'-O-甲氧基乙基修饰的核苷酸。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中第3-9个核苷酸中不超过2个核苷酸为2'-O-甲氧基乙基修饰的核苷酸。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2'-O-甲氧基乙基修饰的核苷酸的话，为2'-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2'-O-甲氧基乙基修饰的核苷酸的话，为2'-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。
在一些实施方式中，本公开的siRNA可以是以下第一种和/或第二种和/或第三种和/或第四种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'-CUAGGAGGCUGUAGGCAUZ 1-3'(SEQ ID NO:1)；
5'-Z 2AUGCCUACAGCCUCCUAG-3'(SEQ ID NO:2),
其中，所述Z 1为A,Z 2为U，所述核苷酸序列I中包含位置对应于Z 1的核苷酸Z 3，所述核苷酸序列II中包含位置对应于Z 2的核苷酸Z 4，所述Z 4是所述反义链5'末端的第一个核苷酸。在一些实施方式中，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2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异。其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间的核苷酸差异可包括Z 3位置处的差异和/或核苷酸序列I中任意一个其它核苷酸位置处的核苷酸差异。在一些实施方式中，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间的核苷酸差异可包括Z 3位置处和/或与Z 3相邻核苷酸位置处的核苷酸差异。
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Z 4位置处的差异，且Z 4选自A、G或C。在一些实施方式中，所述Z 3是与Z 4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Z 4位置处的差异，且Z 4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可任意地被T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，可在保持低的脱靶效应的同时，进一步提升本公开的siRNA的目标基因表达抑制活性。
在一些实施方式中，所述核苷酸序列I是SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:7所示的核苷酸序列：
5'-CUAGGAGGCUGUAGGCAUZ 3-3'(SEQ ID NO:3)；
5'-CUAGGAGGCUGUAGGCACZ 3-3'(SEQ ID NO:4)；
5'-CUAGGAGGCUGUAGGCAGZ 3-3'(SEQ ID NO:5)；
5'-CUAGGAGGCUGUAGGCAAZ 3-3'(SEQ ID NO:6)；
5'-Z 4AUGCCUACAGCCUCCUAG-3'(SEQ ID NO:7),
其中，Z 3选自A、U、G或C，Z 4是与Z 3互补的核苷酸。在一些实施方式中，Z 3为A，Z 4为U。
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，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，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的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，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，所述第二段核苷酸序列是指和HBV基因表达的mRNA中与第一段核苷酸序列相邻、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GUA，所述核苷酸序列IV的碱基组成为UAC，所述第二段核苷酸序列的碱基组成为GUA，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UGUA，所述核苷酸序列IV的碱基组成为UACA，所述第二段核苷酸序列的碱基组成为UGUA，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RNA，其中所述核苷酸序列I与SEQ ID NO:46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:47所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GGCUUUCAGUUAUAUGGAZ 5-3'(SEQ ID NO:46)；
5'-Z 6UCCAUAUAACUGAAAGCC-3'(SEQ ID NO:47),
其中Z 5为U,Z 6为A，所述核苷酸序列I中包含位置对应于Z 5的核苷酸Z 7，所述核苷酸序列II中包含位置对应于Z 6的核苷酸Z 8，所述Z 8是所述反义链5'末端的第一个核苷酸。在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:46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:46所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:47所示的核苷酸序列之间不多于1个核苷酸差异。其中，所述核苷酸序列I与SEQ ID NO:46所示的核苷酸序列之间的核苷酸差异可包括Z 7位置处的差异和/或核苷酸序列I中任意一个其它核苷酸位置处的核苷酸差异。在一些实施方式中，其中，所述核苷酸序列I与SEQ ID NO:46所示的核苷酸序列之间的核苷酸差异可包括Z 7位置处和/或与Z 7相邻核苷酸位置处的核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:47所示的核苷酸序列之间的差异包括Z 8位置处的差异，且Z 8选自U、G或C。在一些实施方式中，所述Z 7是与Z 8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:47所示的核苷酸序列之间的差异为Z 8位置处的差异，且Z 8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可任意地被T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，可在保持低的脱靶效应的同时，进一步提升本公开的siRNA的目标基因表达抑制活性。
在一些实施方式中，核苷酸序列I是SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:52所示的核苷酸序列：
5'-GGCUUUCAGUUAUAUGGAZ 7-3'(SEQ ID NO:48)；
5'-GGCUUUCAGUUAUAUGGCZ 7-3'(SEQ ID NO:49)；
5'-GGCUUUCAGUUAUAUGGGZ 7-3'(SEQ ID NO:50)；
5'-GGCUUUCAGUUAUAUGGUZ 7-3'(SEQ ID NO:51)；
5'-Z 8UCCAUAUAACUGAAAGCC-3'(SEQ ID NO:52),
其中，Z 7选自A、U、G或C，Z 8是与Z 7互补的核苷酸。在一些实施方式中，Z 7为U，Z 8为A。
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，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，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的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，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，所述第二段核苷酸序列是指和HBV基因表达的mRNA中与第一段核苷酸序列相邻、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I与SEQ ID NO:46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U，所述核苷酸序列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UU，所述核苷酸序列IV的碱基组成为AA，所述第二段核苷酸序列的碱基组成为UU；此时，正义链和反义链的长度比为21/21；或者，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UUU，所述核苷酸序列IV的碱基组成为AAA，所述第二段核苷酸序列的碱基组成为UUU，此时，正义链和反义链的长度比为22/22；或者，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GUUU，所述核苷酸序列IV的碱基组成为AAAC，所述第二段核苷酸序列的碱基组成为GUUU；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:91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:92所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-CCUUGAGGCAUACUUCAAZ 9-3'(SEQ ID NO:91)；
5'-Z 10UUGAAGUAUGCCUCAAGG-3'(SEQ ID NO:92),
其中Z 9为A,Z 10为U，所述核苷酸序列I中包含位置对应于Z 9的核苷酸Z 11，所述核苷酸序列II中包含位置对应于Z 10的核苷酸Z 12，所述Z 12是所述反义链5'末端的第一个核苷酸。在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:91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:91所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:92所示的核苷酸序列之间不多于1个核苷酸差异。其中，所述核苷酸序列I与SEQ ID NO:91所示的核苷酸序列之间的核苷酸差异可包括Z 11位置处的差异和/或核苷酸序列I中任意一个其它核苷酸位置处的核苷酸差异。在一些实施方式中，其中，所述核苷酸序列I与SEQ ID NO:91所示的核苷酸序列之间的核苷酸差异可包括Z 11位置处和/或与Z 11相邻核苷酸位置处的核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:92所示的核苷酸序列之间的差异包括Z 12位置处的差异，且Z 12选自A、G或C。在一些实施方式中，所述Z 11是与Z 12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:92所示的核苷酸序列之间的差异为Z 12位置处的差异，且Z 12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可任意地被T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，可在保持低的脱靶效应的同时，进一步提升本公开的siRNA的目标基因表达抑制活性。
在一些实施方式中，核苷酸序列I是SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:97所示的核苷酸序列：
5'-CCUUGAGGCAUACUUCAAZ 11-3'(SEQ ID NO:93)；
5'-CCUUGAGGCAUACUUCAUZ 11-3'(SEQ ID NO:94)；
5'-CCUUGAGGCAUACUUCAGZ 11-3'(SEQ ID NO:95)；
5'-CCUUGAGGCAUACUUCACZ 11-3'(SEQ ID NO:96)；
5'-Z 12UUGAAGUAUGCCUCAAGG-3'(SEQ ID NO:97),
其中，Z 11选自A、U、G或C，Z 12是与Z 11互补的核苷酸。在一些实施方式中，Z 11为A，Z 12为U。
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，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，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的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，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，所述第二段核苷酸序列是指和HBV基因表达的mRNA中与第一段核苷酸序列相邻、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I与SEQ ID NO:91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GA，所述核苷酸序列IV的碱基组成为UC，所述第二段核苷酸序列的碱基组成为GA；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CGA，所述核苷酸序列IV的碱基组成为UCG，所述第二段核苷酸序列的碱基组成为CGA，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CCGA，所述核苷酸序列IV的碱基组成为UCGG，所述第二段核苷酸序列的碱基组成为CCGA；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:136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:137所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-UGCUAUGCCUCAUCUUCUZ 13-3'(SEQ ID NO:136)；
5'-Z 14AGAAGAUGAGGCAUAGCA-3'(SEQ ID NO:137),
其中Z 13为A,Z 14为U，所述核苷酸序列I中包含位置对应于Z 13的核苷酸Z 15，所述核苷酸序列II中包含位置对应于Z 14的核苷酸Z 16，所述Z 16是所述反义链5'末端的第一个核苷酸。在一些实施方式中，所述第一段核苷酸序列是如SEQ ID NO:136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:136所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:137所示的核苷酸序列之间不多于1个核苷酸差异。其中，其中，所述核苷酸序列I与SEQ ID NO:136所示的核苷酸序列之间的核苷酸差异可包括Z 15位置处的差异和/或核苷酸序列I中任意一个其它核苷酸位置处的核苷酸差异。在一些实施方式中，其中，所述核苷酸序列I与SEQ ID NO:136所示的核苷酸序列之间的核苷酸差异可包括Z 15位置处和/或与Z 15相邻核苷酸位置处的核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:137所示的核苷酸序列之间的差异包括Z 16位置处的差异，且Z 16选自A、G或C。在一些实施方式中，所述Z 15是与Z 16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:137所示的核苷酸序列之间的差异为Z 16位置处的差异，且Z 16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可任意地被T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，可在保持低的脱靶效应的同时，进一步提升本公开的siRNA的目标基因表达抑制活性。
在一些实施方式中，核苷酸序列I是SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140或SEQ ID NO:141所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:142所示的核苷酸序列：
5'-UGCUAUGCCUCAUCUUCUZ 15-3'(SEQ ID NO:138)；
5'-UGCUAUGCCUCAUCUUCAZ 15-3'(SEQ ID NO:139)；
5'-UGCUAUGCCUCAUCUUCGZ 15-3'(SEQ ID NO:140)；
5'-UGCUAUGCCUCAUCUUCCZ 15-3'(SEQ ID NO:141)；
5'-Z 16AGAAGAUGAGGCAUAGCA-3'(SEQ ID NO:142),
其中，Z 15选自A、U、G或C，Z 16是与Z 15互补的核苷酸。在一些实施 方式中，Z 15为A，Z 16为U。
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，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，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的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，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，所述第二段核苷酸序列是指和HBV基因表达的mRNA中与第一段核苷酸序列相邻、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I与SEQ ID NO:136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C，所述核苷酸序列IV的碱基为G，所述第二段核苷酸序列的碱基为C；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GC，所述核苷酸序列IV的碱基组成为GC，所述第二段核苷酸序列的碱基组成为GC；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UGC，所述核苷酸序列IV的碱基组成为GCA，所述第二段核苷酸序列的碱基组成为UGC，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CUGC，所述核苷酸序列IV的碱基组成为GCAG，所述第二段核苷酸序列的碱基组成为CUGC；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。
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和第四种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siRNA，例如对第一种siRNA、第二种siRNA、第三种siRNA和第四种siRNA逐一进行了描述。例如，如不特别指明具体的siRNA，“所述siRNA还含有核苷酸序列V”的意思是“本公开的siRNA，例如上述第一种siRNA、第二种siRNA、第三种siRNA或第四种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，所述核苷酸序列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(例如，不是2'-O-甲氧基乙基修饰的核苷酸)，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，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(dTdT)、连续的2个尿嘧啶核糖核苷酸(UU)；或者，为了提高siRNA反义链与靶mRNA的亲和力，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HBV基因表达的mRNA中与第一段核苷酸序列或第二段核苷酸序列相邻、并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UA。所述siRNA的正义链含有如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，所述反义链含有如SEQ ID NO:8所示的核苷酸序列：
5'-CUAGGAGGCUGUAGGCAUZ 3-3'(SEQ ID NO:3)；
5'-CUAGGAGGCUGUAGGCACZ 3-3'(SEQ ID NO:4)；
5'-CUAGGAGGCUGUAGGCAGZ 3-3'(SEQ ID NO:5)；
5'-CUAGGAGGCUGUAGGCAAZ 3-3'(SEQ ID NO:6)；
5'-Z 4AUGCCUACAGCCUCCUAGUA-3'(SEQ ID NO:8),
其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C，并且Z 4是与Z 3互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:9、SEQ ID NO:10、SEQ ID  NO:11或SEQ ID NO:12所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:13所示的核苷酸序列：
5'-UACUAGGAGGCUGUAGGCAUZ 3-3'(SEQ ID NO:9)；
5'-UACUAGGAGGCUGUAGGCACZ 3-3'(SEQ ID NO:10)；
5'-UACUAGGAGGCUGUAGGCAGZ 3-3'(SEQ ID NO:11)；
5'-UACUAGGAGGCUGUAGGCAAZ 3-3'(SEQ ID NO:12)；
5'-Z 4AUGCCUACAGCCUCCUAGUACA-3'(SEQ ID NO:13),
其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C，并且Z 4是与Z 3互补的核苷酸。
在一些实施方式中，对于所述第二种siRNA，所述第一段核苷酸序列具有SEQ ID NO:46所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是UU。所述siRNA的正义链含有如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:53所示的核苷酸序列：
5'-GGCUUUCAGUUAUAUGGAZ 7-3'(SEQ ID NO:48)；
5'-GGCUUUCAGUUAUAUGGCZ 7-3'(SEQ ID NO:49)；
5'-GGCUUUCAGUUAUAUGGGZ 7-3'(SEQ ID NO:50)；
5'-GGCUUUCAGUUAUAUGGUZ 7-3'(SEQ ID NO:51)；
5'-Z 8UCCAUAUAACUGAAAGCCAA-3'(SEQ ID NO:53),
其中，所述Z 8是反义链5'末端的第一个核苷酸，Z 7选自A、U、G或C，并且Z 8是与Z 7互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或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'-UUGGCUUUCAGUUAUAUGGAZ 7-3'(SEQ ID NO:54)；
5'-UUGGCUUUCAGUUAUAUGGCZ 7-3'(SEQ ID NO:55)；
5'-UUGGCUUUCAGUUAUAUGGGZ 7-3'(SEQ ID NO:56)；
5'-UUGGCUUUCAGUUAUAUGGUZ 7-3'(SEQ ID NO:57)；
5'-Z 8UCCAUAUAACUGAAAGCCAAAC-3'(SEQ ID NO:58),
其中，所述Z 8是反义链5'末端的第一个核苷酸，Z 7选自A、U、G或C，并且Z 8是与Z 7互补的核苷酸。
在一些实施方式中，对于所述第三种siRNA，所述第一段核苷酸序列具有SEQ ID NO:91所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是GA。所述siRNA的正义链含有如SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:98所示的核苷酸序列：
5'-CCUUGAGGCAUACUUCAAZ 11-3'(SEQ ID NO:93)；
5'-CCUUGAGGCAUACUUCAUZ 11-3'(SEQ ID NO:94)；
5'-CCUUGAGGCAUACUUCAGZ 11-3'(SEQ ID NO:95)；
5'-CCUUGAGGCAUACUUCACZ 11-3'(SEQ ID NO:96)；
5'-Z 12UUGAAGUAUGCCUCAAGGUC-3'(SEQ ID NO:98),
其中，所述Z 12是反义链5'末端的第一个核苷酸，Z 11选自A、U、G或C，并且Z 12是与Z 11互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101或SEQ ID NO:102所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:103所示的核苷酸序列：
5'-GACCUUGAGGCAUACUUCAAZ 11-3'(SEQ ID NO:99)；
5'-GACCUUGAGGCAUACUUCAUZ 11-3'(SEQ ID NO:100)；
5'-GACCUUGAGGCAUACUUCAGZ 11-3'(SEQ ID NO:101)；
5'-GACCUUGAGGCAUACUUCACZ 11-3'(SEQ ID NO:102)；
5'-Z 12UUGAAGUAUGCCUCAAGGUCGG-3'(SEQ ID NO:103),
其中，所述Z 12是反义链5'末端的第一个核苷酸，Z 11选自A、U、G或C，并且Z 12是与Z 11互补的核苷酸。
在一些实施方式中，对于所述第四种siRNA，所述第一段核苷酸序列具有SEQ ID NO:136所示的核苷酸序列，所述第三段核苷酸序列的碱基组成是GC。所述siRNA的正义链含有如SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140或SEQ ID NO:141所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:143所示的核苷酸序列：
5'-UGCUAUGCCUCAUCUUCUZ 15-3'(SEQ ID NO:138)；
5'-UGCUAUGCCUCAUCUUCAZ 15-3'(SEQ ID NO:139)；
5'-UGCUAUGCCUCAUCUUCGZ 15-3'(SEQ ID NO:140)；
5'-UGCUAUGCCUCAUCUUCCZ 15-3'(SEQ ID NO:141)；
5'-Z 16AGAAGAUGAGGCAUAGCAGC-3'(SEQ ID NO:143),
其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 15选自A、U、G或C，并且Z 16是与Z 15互补的核苷酸；
或者，所述siRNA的正义链含有如SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146或SEQ ID NO:147所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:148所示的核苷酸序列：
5'-GCUGCUAUGCCUCAUCUUCUZ 15-3'(SEQ ID NO:144)；
5'-GCUGCUAUGCCUCAUCUUCAZ 15-3'(SEQ ID NO:145)；
5'-GCUGCUAUGCCUCAUCUUCGZ 15-3'(SEQ ID NO:146)；
5'-GCUGCUAUGCCUCAUCUUCCZ 15-3'(SEQ ID NO:147)；
5'-Z 16AGAAGAUGAGGCAUAGCAGCAG-3'(SEQ ID NO:148),
其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 15选自A、U、G或C，并且Z 16是与Z 15互补的核苷酸。
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HBV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2'-氟代修饰的核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，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2'-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。
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2'-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2'-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。
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'-NH 2)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：
Figure PCTCN2022140698-appb-000001
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3'-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2'-、4'-位处以提供一个2',4'-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12)所示，ENA如式(13)所示，cET BNA如式(14)所示：
Figure PCTCN2022140698-appb-000002
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中，无环核苷酸可以是解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(15)所示，GNA如式(16)所示：
Figure PCTCN2022140698-appb-000003
上述式(15)和式(16)中，R选自H、OH或烷氧基(O-烷基)。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方式中，异核苷酸可以是碱基从核糖环的1'-位移动至2'-位或3'-位而形成的化合物，如式(17)或(18)所示。
Figure PCTCN2022140698-appb-000004
上述式(17)-式(18)化合物中，Base表示核酸碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET BNA、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”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”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；按照5'末端到3'末端的方向，在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者第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糖核酸酶水解的性能。同时，上述修饰降低了siRNA的脱靶效应并未显著降低siRNA的抑制性能。
在一些实施方式中，本公开提供的siRNA为表1a-1d中列出的siHBVa1-M1、siHBVa1-M2、siHBVa2-M1、siHBVa2-M2、siHBVa3-M1、siHBVa3-M2、siHBVa4-M1、siHBVa4-M2、siHBVa5-M1、siHBVa5-M2、siHBVa6-M1、siHBVa6-M2、siHBVa7-M1、siHBVa7-M2、siHBVa8-M1、siHBVa8-M2、siHBVb1-M1、siHBVb1-M2、siHBVb2-M1、siHBVb2-M2、siHBVb3-M1、siHBVb3-M2、 siHBVb4-M1、siHBVb4-M2、siHBVb5-M1、siHBVb5-M2、siHBVb6-M1、siHBVb6-M2、siHBVb7-M1、siHBVb7-M2、siHBVb8-M1、siHBVb8-M2、siHBVc1-M1、siHBVc1-M2、siHBVc2-M1、siHBVc2-M2、siHBVc3-M1、siHBVc3-M2、siHBVc4-M1、siHBVc4-M2、siHBVc5-M1、siHBVc5-M2、siHBVc6-M1、siHBVc6-M2、siHBVc7-M1、siHBVc7-M2、siHBVc8-M1、siHBVc8-M2、siHBVd1-M1、siHBVd1-M2、siHBVd2-M1、siHBVd2-M2、siHBVd3-M1、siHBVd3-M2、siHBVd4-M1、siHBVd4-M2、siHBVd5-M1、siHBVd5-M2、siHBVd6-M1、siHBVd6-M2、siHBVd7-M1、siHBVd7-M2、siHBVd8-M1或siHBVd8-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亚基；在一些实施方式中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯亚基：
Figure PCTCN2022140698-appb-000005
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。
在一些实施方式中，本公开提供的siRNA中，硫代磷酸酯亚基连接存在于由以下位置组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯亚基连接存在于除正义链5'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯亚基连接存在于除正义链3'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯亚基连接存在于以下位置中的至少一处：
所述正义链的5'末端端部第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-1d中列出的siHBVa1-M1S、siHBVa1-M2S、siHBVa2-M1S、siHBVa2-M2S、siHBVa3-M1S、siHBVa3-M2S、siHBVa4-M1S、siHBVa4-M2S、siHBVa5-M1S、siHBVa5-M2S、siHBVa6-M1S、siHBVa6-M2S、siHBVa7-M1S、siHBVa7-M2S、siHBVa8-M1S、siHBVa8-M2S、siHBVb1-M1S、siHBVb1-M2S、siHBVb2-M1S、siHBVb2-M2S、siHBVb3-M1S、siHBVb3-M2S、siHBVb4-M1S、siHBVb4-M2S、siHBVb5-M1S、siHBVb5-M2S、siHBVb6-M1S、siHBVb6-M2S、siHBVb7-M1S、siHBVb7-M2S、siHBVb8-M1S、siHBVb8-M2S、siHBVc1-M1S、siHBVc1-M2S、siHBVc2-M1S、siHBVc2-M2S、siHBVc3-M1S、siHBVc3-M2S、siHBVc4-M1S、siHBVc4-M2S、siHBVc5-M1S、siHBVc5-M2S、siHBVc6-M1S、siHBVc6-M2S、siHBVc7-M1S、siHBVc7-M2S、siHBVc8-M1S、siHBVc8-M2S、siHBVd1-M1S、siHBVd1-M2S、siHBVd2-M1S、siHBVd2-M2S、siHBVd3-M1S、siHBVd3-M2S、siHBVd4-M1S、siHBVd4-M2S、siHBVd5-M1S、siHBVd5-M2S、siHBVd6-M1S、siHBVd6-M2S、siHBVd7-M1S、siHBVd7-M2S、siHBVd8-M1S或siHBVd8-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：
Figure PCTCN2022140698-appb-000006
再如，Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48中公开了如下4种5'-磷酸类似物修饰的核苷酸：
Figure PCTCN2022140698-appb-000007
其中，R选自H、OH、甲氧基、氟；Base表示核酸碱基，选自A、U、C、G或T。
在一些实施方式中，5'-磷酸核苷酸为式(2)所示的含有5'-磷酸修饰的核苷酸，5'-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5'-(E)- vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者为硫代磷酸酯修饰的核苷酸，如式(5)所示。
在一些实施方式中，本公开的siRNA为下表1a-1d中列出的siHBVa1-M1P1、siHBVa1-M2P1、siHBVa2-M1P1、siHBVa2-M2P1、siHBVa3-M1P1、siHBVa3-M2P1、siHBVa4-M1P1、siHBVa4-M2P1、siHBVa5-M1P1、siHBVa5-M2P1、siHBVa6-M1P1、siHBVa6-M2P1、siHBVa7-M1P1、siHBVa7-M2P1、siHBVa8-M1P1、siHBVa8-M2P1、siHBVa1-M1SP1、siHBVa1-M2SP1、siHBVa2-M1SP1、siHBVa2-M2SP1、siHBVa3-M1SP1、siHBVa3-M2SP1、siHBVa4-M1SP1、siHBVa4-M2SP1、siHBVa5-M1SP1、siHBVa5-M2SP1、siHBVa6-M1SP1、siHBVa6-M2SP1、siHBVa7-M1SP1、siHBVa7-M2SP1、siHBVa8-M1SP1、siHBVa8-M2SP1、siHBVb1-M1P1、siHBVb1-M2P1、siHBVb2-M1P1、siHBVb2-M2P1、siHBVb3-M1P1、siHBVb3-M2P1、siHBVb4-M1P1、siHBVb4-M2P1、siHBVb5-M1P1、siHBVb5-M2P1、siHBVb6-M1P1、siHBVb6-M2P1、siHBVb7-M1P1、siHBVb7-M2P1、siHBVb8-M1P1、siHBVb8-M2P1、siHBVb1-M1SP1、siHBVb1-M2SP1、siHBVb2-M1SP1、siHBVb2-M2SP1、siHBVb3-M1SP1、siHBVb3-M2SP1、siHBVb4-M1SP1、siHBVb4-M2SP1、siHBVb5-M1SP1、siHBVb5-M2SP1、siHBVb6-M1SP1、siHBVb6-M2SP1、siHBVb7-M1SP1、siHBVb7-M2SP1、siHBVb8-M1SP1、siHBVb8-M2SP1、siHBVc1-M1P1、siHBVc1-M2P1、siHBVc2-M1P1、siHBVc2-M2P1、siHBVc3-M1P1、siHBVc3-M2P1、siHBVc4-M1P1、siHBVc4-M2P1、siHBVc5-M1P1、siHBVc5-M2P1、siHBVc6-M1P1、siHBVc6-M2P1、siHBVc7-M1P1、siHBVc7-M2P1、siHBVc8-M1P1、siHBVc8-M2P1、siHBVc1-M1SP1、siHBVc1-M2SP1、siHBVc2-M1SP1、siHBVc2-M2SP1、siHBVc3-M1SP1、siHBVc3-M2SP1、siHBVc4-M1SP1、siHBVc4-M2SP1、siHBVc5-M1SP1、siHBVc5-M2SP1、siHBVc6-M1SP1、siHBVc6-M2SP1、siHBVc7-M1SP1、siHBVc7-M2SP1、siHBVc8-M1SP1、siHBVc8-M2SP1、siHBVd1-M1P1、siHBVd1-M2P1、siHBVd2-M1P1、siHBVd2-M2P1、siHBVd3-M1P1、siHBVd3-M2P1、siHBVd4-M1P1、siHBVd4-M2P1、siHBVd5-M1P1、siHBVd5-M2P1、siHBVd6-M1P1、siHBVd6-M2P1、siHBVd7-M1P1、siHBVd7-M2P1、siHBVd8-M1P1、siHBVd8-M2P1、siHBVd1-M1SP1、siHBVd1-M2SP1、siHBVd2-M1SP1、siHBVd2-M2SP1、siHBVd3-M1SP1、siHBVd3-M2SP1、siHBVd4-M1SP1、siHBVd4-M2SP1、siHBVd5-M1SP1、siHBVd5-M2SP1、siHBVd6-M1SP1、siHBVd6-M2SP1、siHBVd7-M1SP1、siHBVd7-M2SP1、siHBVd8-M1SP1或siHBVd8-M2SP1中的一种。
本公开的发明人意外发现，本公开提供的siRNA不仅具有显著增强的血浆和溶酶体稳定性、显著低的脱靶效应，还保留很高的基因抑制活性。
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以 通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
siRNA缀合物
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连接或非共价连接，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，“缀合基团”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和任选的接头(linker)，并且，所述siRNA、所述接头和所述靶向基团依次连接。在一些实施方式中，所述靶向基团为1-6个。在一些实施方式中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在siRNA正义链的3'端或5'端，也可在反义链的5'端，还可以在siRNA的内部序列中。在一些实施方式中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3'末端。
在一些实施方式中，所述缀合基团可以连接在核苷酸的磷酸基团、2'-位羟基或者碱基上。在一些实施方式中，所述缀合基团还可以连接在3'-位羟基上，此时核苷酸之间采用2'-5'磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology,2015,10(5):1181-7。
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HBV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胺或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：
Figure PCTCN2022140698-appb-000008
其中，k为1-3的整数；
L A具有如式(302)所示的包含酰胺键的结构，L B具有如式(303)所示的 包含N-酰基吡咯烷的结构，含有羰基和氧原子，L C为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团；
Figure PCTCN2022140698-appb-000009
其中，n 302、q 302和p 302各自独立地为2-6的整数，可选地，n 302、q 302和p 302各自独立地为2或3；n 303为4-16的整数，可选地，n 303为8-12的整数，
Figure PCTCN2022140698-appb-000010
表示基团共价连接的位点。
所述接头中，每个L A分别与一个所述靶向基团通过醚键连接，并通过L C部分中羟基的氧原子与L C部分形成醚键而连接；L B通过式(303)中的羰基与L C部分中氨基的氮原子形成酰胺键而连接，并通过式(303)中的氧原子与所述siRNA通过氧原子形成磷酸酯键或硫代磷酸酯键相连接。
在一些实施方式中，本公开提供的siRNA缀合物具有如式(305)所示的结构：
Figure PCTCN2022140698-appb-000011
其中，Nu表示本公开提供的siRNA。
在一些实施方式中，本公开的siRNA缀合物中的接头具有式(306)所示的结构：
Figure PCTCN2022140698-appb-000012
其中，n 306为0-3的整数，每个p 306独立地为1-6的整数，
Figure PCTCN2022140698-appb-000013
表示基团共价连接的位点；所述连接基团通过由*标出的氧原子与所述靶向基团形成醚键连接；所述连接基团由#标出的氧原子中的至少一个与所述siRNA形成磷酸酯键或硫代磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C 1-C 3烷基连接形成C 1-C 3烷氧基；
在一些实施方式中，本公开的siRNA缀合物具有如式(307)所示的结构：
Figure PCTCN2022140698-appb-000014
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：
Figure PCTCN2022140698-appb-000015
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；
R 10、R 11、R 12、R 13、R 14或R 15各自独立地为H，或选自于由以下基团所组成的组：C 1-C 10烷基、C 1-C 10卤代烷基以及C 1-C 10烷氧基；
R 3具有式A59所示的结构：
Figure PCTCN2022140698-appb-000016
其中，E 1为OH、SH或BH 2，Nu表示本公开提供的siRNA；
R 2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，R 2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2、-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
每个L 1独立地是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，L 1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2，-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1- C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
Figure PCTCN2022140698-appb-000017
表示基团共价连接的位点；
M 1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M 1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。
技术人员会理解的是，尽管为了方便起见，L 1被定义为线性烷基，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，L 1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M 1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体时，在一些实施方式中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述缀合物中M 1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M 1配体的个数至少为3，使得M 1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M 1配体的个数大于3个时，M 1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M 1配体之间的空间位置适合M 1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R 10、R 11、R 12、R 13、R 14和R 15各自独立地选自H、C 1-C 10烷基、C 1-C 10卤代烷基、以及C 1-C 10烷氧基中的一种时，不会改变本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，R 10、R 11、R 12、R 13、R 14和R 15各自独立地选自H、甲基和乙基。在一些实施方式中，R 10、R 11、R 12、R 13、R 14和R 15均为H。
根据本公开提供的siRNA缀合物，R 3为式A59所示结构的基团，其中，E 1为OH、SH或BH 2，基于制备原料易获取性的考虑，在一些实施方式中，E 1为OH或SH。
在一些实施方式中，R 2的选择是为了实现含氮骨架上的N与A59的连接。 在本公开的上下文中，“含氮骨架”是指连接有R 10、R 11、R 12、R 13、R 14和R 15的碳原子与N互相连接的链状结构。因此，R 2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N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，R 2基团中需要同时含有与含氮骨架上的N连接的连接位点和与R 3中的P相连接的连接位点。在一些实施方式中，R 2中所述与含氮骨架上的N连接的位点与N形成酰胺键，所述与R 3上的P连接的位点与P形成磷酸酯键。在一些实施方式中，R 2的长度为4-15个原子。在一些实施方式中，R 2是B5、B6、B5’或B6’：
Figure PCTCN2022140698-appb-000018
其中，
Figure PCTCN2022140698-appb-000019
表示基团共价键连接的位点。
q 2的取值范围可以是1-10的整数，在一些实施方式中，q 2为1-5的整数。
L 1的作用是将M 1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，L 1选自式A1-A26基团中的一种或多种的连接组合。在一些实施方式中，L 1选自A1、A4、A5、A6、A8、A10、A11和A13中的一种或多种的连接组合；在一些实施方式中，L 1选自A1、A4、A8、A10和A11中至少2个的连接组合；在一些实施方式中，L 1选自A1、A8、A10中至少2个的连接组合。
Figure PCTCN2022140698-appb-000020
Figure PCTCN2022140698-appb-000021
在一些实施方式中，L 1的长度可以为3-25个原子，3-20个原子、4-15个原子或5-12个原子。在一些实施方式中是，L 1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。
在一些实施方式中，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C 1-C 4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C 1-C 5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M 1配体与含氮骨架上的N连接，并使M 1配体之间的空间位置更适合M 1配体与肝表面去唾液酸糖蛋白受体结合。
Figure PCTCN2022140698-appb-000022
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：
Figure PCTCN2022140698-appb-000023
Figure PCTCN2022140698-appb-000024
Figure PCTCN2022140698-appb-000025
Figure PCTCN2022140698-appb-000026
Figure PCTCN2022140698-appb-000027
Figure PCTCN2022140698-appb-000028
Figure PCTCN2022140698-appb-000029
其中，Nu表示本公开的siRNA。
在一些实施方式中，式A59中的P可以连接到siRNA序列中任何可能的位置，例如，式A59中的P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。
在一些实施方式中，本公开的siRNA缀合物包含的siRNA可以是例如表1a、表1b、表1c或表1d中列出的任何一种siRNA。包含这些siRNA的siRNA缀合物表现出低的脱靶效应和高的HBV基因表达的mRNA抑制活性。
表1a本公开的第一种siRNA序列
Figure PCTCN2022140698-appb-000030
Figure PCTCN2022140698-appb-000031
Figure PCTCN2022140698-appb-000032
Figure PCTCN2022140698-appb-000033
表1b本公开的第二种siRNA序列
Figure PCTCN2022140698-appb-000034
Figure PCTCN2022140698-appb-000035
Figure PCTCN2022140698-appb-000036
Figure PCTCN2022140698-appb-000037
表1c本公开的第三种siRNA序列
Figure PCTCN2022140698-appb-000038
Figure PCTCN2022140698-appb-000039
Figure PCTCN2022140698-appb-000040
Figure PCTCN2022140698-appb-000041
表1d本公开的第四种siRNA序列
Figure PCTCN2022140698-appb-000042
Figure PCTCN2022140698-appb-000043
Figure PCTCN2022140698-appb-000044
Figure PCTCN2022140698-appb-000045
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亚基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。在一些实施方式中， 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，每个 S均是 moe。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-1d中所列的序列中的每个U可任意地被T替换，不会对siRNA的活性或脱靶效应产生明显影响。
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， 和/或如上所述的siRNA缀合物作为活性成分和药学上可接受的载体。
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe 3O 4或Fe 2O 3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
在一些实施方式中，所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，在一些实施方式中，siRNA与药学上可接受的载体的重量比可以为1:(1-500)。在一些的实施方式中，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)、或通过口咽吸入、或鼻腔给药等方式递送所述药物组合物。在一些实施方式中，所述药物组合物用于喷雾给药。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于中国专利申请CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
在一些实施方式中，所述有机胺可为中国专利申请CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
Figure PCTCN2022140698-appb-000046
其中：
X 101和X 102各自独立地是O、S、N-A或C-A，其中A是氢或C 1-C 20烃链；
Y 101和Z 101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO 2；
R 101、R 102、R 103、R 104、R 105、R 106和R 107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R 102是氢；
并且，如果n或m中的至少一个是2，那么R 103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
Figure PCTCN2022140698-appb-000047
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。
在一些实施方式中，R 103是多胺。在其它实施方式中，R 103是缩酮。在一些实施方式中，在式(201)中的R 101和R 102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R 103可以是下述式(204)-式(213)中的任一个：
Figure PCTCN2022140698-appb-000048
Figure PCTCN2022140698-appb-000049
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R 103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
其中，式(201)所示化合物可以根据中国专利申请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：
Figure PCTCN2022140698-appb-000050
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pH＝7.4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。
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知的方法制备。例如，WO2015006740A2中详细描述了多种siRNA缀合物的制备方法，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。，此外，可以通过本领域技术人员熟知的其它方式，获得本公开的siRNA缀合物。
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HBV基因表达的mRNA水平相关的疾病或者症状的药物中的用途。在一些实施方式中，所述与HBV基因表达的mRNA水平相关的疾病或者症状是慢性肝病、炎症、纤维化疾病和增生性疾病中的至少一种。
在一些实施方式中，本公开提供了一种治疗和/或预防与HBV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HBV基因表达的mRNA水平相关的疾病或症状是慢性肝病、炎症、纤维化疾病和增生性疾病中的至少一种。
在一些实施方式中，本公开还提供了一种抑制细胞中HBV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/或药物组合物和/或siRN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/或治疗由细胞中特 定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/或治疗由肝细胞中特定基因的表达而引起的病理状况或疾病的手段，在一些实施方式中为能够将药物递送至肝脏的给药方式。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年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％的群体致死的剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中，指引起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基团形成的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缀合物导入特定基因异常表达的细胞，还可以通过基因表达调控的机制达到抑制细胞中该特定基因的表达这一目的。在一些实施方式中，所述细胞为肝细胞。在一些实施方式中，所述肝细胞可以是选自Hep3B、HepG2、Huh7等肝癌细胞系的细胞或分 离的原代肝细胞，在一些实施方式中为原代肝细胞。
采用本公开提供的方法抑制特定基因在细胞中表达，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，所述试剂盒包含本公开提供的siRNA、和/或药物组合物和/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 LBboratory Press(1989))所记载的方法进行。
制备例1-16本公开提供的siRNA缀合物的合成
按照CN110959011A制备例14所述的制备方法，制备获得了以下表2中的缀合物1-16，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16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1-16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基团分别具有表2中缀合物1-16所对应的siRNA序列，并且所述siRNA缀合物处于钠盐形式。
表2 siRNA缀合物中的siRNA序列
Figure PCTCN2022140698-appb-000051
Figure PCTCN2022140698-appb-000052
Figure PCTCN2022140698-appb-000053
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；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯亚基连接；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；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。
对比制备例1-10参比siRNA缀合物的合成
按照CN110959011A制备例14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1-9和参比缀合物NC，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1-9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1-9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1-9和参比缀合物NC所对应的siRNA序列，这些siRNA序列中不包含稳定化修饰核苷酸。
对于上述制备例制备得到的缀合物和参比缀合物，本公开的发明人利用如下方法测定了其双链热解离温度Tm，测试方法如下：
用1×PBS缓冲液将上述制备的缀合物11和参比缀合物9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3所示：
表3双链热解离温度Tm
缀合物 Tm(℃) ΔTm(℃)
参比缀合物9 65.02 0
缀合物11 66.07 1.05
其中，
ΔTm值(待测缀合物)＝Tm(待测缀合物)-Tm(参比缀合物9)；
根据表3的结果可知，，与相同位置为未修饰的核苷酸的情况相比，本公开的包含稳定化修饰核苷酸的双链寡核苷酸及其缀合物具有更高的双链热解离温度。
实验例1siRNA缀合物在体外psi-CHECK系统中的抑制活性
本实验例中，采用体外psi-CHECK系统，检测了缀合物1、缀合物2、缀合物3、缀合物4和参比缀合物1在体外psi-CHECK系统中对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psiCHECK TM-2(Promega TM)质粒构建了检测质粒，所述质粒中含有一个目标序列1，即siRNA缀合物靶序列。对于待测siRNA缀合物，目标序列1如下所示：
5’-CTAGGAGGCTGTAGGCATA-3’(SEQ ID NO:233)
该目标序列1是所检测的siRNA缀合物中反义链的完全互补序列，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psiCHECK TM-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％CO 2/95％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8×10 3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100μL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4μM、1μM、0.25μM、0.0625μM、0.015625μM、0.003906μM、0.0009765μM、0.0002441μM、0.00006104μM、0.00001526μM和0.000003815μM(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3、缀合物4和参比缀合物1。
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 Lipofectamine TM2000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9.95μL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1X12。
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1X12，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缀合物的共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CO 2培养箱继续培养24小时。
[3]检测
吸去培养孔中的培养基，每孔加入150μL的
Figure PCTCN2022140698-appb-000054
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60μL
Figure PCTCN2022140698-appb-000055
Stop&
Figure PCTCN2022140698-appb-000056
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IC 50值，所述函数如下，
Figure PCTCN2022140698-appb-000057
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X 50值，计算获得各siRNA的IC 50值＝10^X 50(nM)，IC 50值总结于表4中。
表4 siRNA缀合物在psi-CHECK系统中的IC 50
缀合物编号 IC 50/(GSCM)
缀合物1 0.0327nM
缀合物2 0.0279nM
缀合物3 0.0278nM
缀合物4 0.0203nM
参比缀合物1 0.0464nM
缀合物1-4和参比缀合物1之间的区别在于：按照5'末端到3'末端的方向，缀合物1正义链的第18个核苷酸上的碱基为C，反义链的第三个核苷酸上具有2'-O-甲氧基乙基修饰基团；缀合物2正义链的第18个核苷酸上的碱基为G，反义链的第三个核苷酸上具有2'-O-甲氧基乙基修饰基团；缀合物3正义链的第18个核苷酸上的碱基为C，反义链的第五个核苷酸上具有2'-O-甲氧基乙基修饰基团；缀合物4正义链的第18个核苷酸上的碱基为G，反义链的第五个核苷酸上具有2'-O-甲氧基乙基修饰基团；参比缀合物1正义链的第18个核苷酸上的碱基为U，反义链上没有2'-O-甲氧基乙基修饰基团。
由上述表4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，IC 50在0.01-0.05nM之间。同时，本公开的siRNA缀合物与不包含稳定化修饰核苷酸的参比缀合物1相比具有相当的目标序列抑制活性，甚至具有比参比缀合物1更高的目标序列抑制活性。
实验例2 siRNA缀合物在体外psi-CHECK系统中的抑制活性
按照实验例1的方法，测试了缀合物1、缀合物2、缀合物3、缀合物4和参比缀合物1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1-4和参比缀合物1使用如下所示的目标序列2代替目标序列1：
目标序列2：5’-GCAGCTTCTTAGGTAGGCATATTGGGCAGCTTCTTAGGTAGGCATATTGGGCAGCTTCTTAGGTAGGCATATTGGGCAGCTTCTTAGGTAGGCATA-3’(SEQ ID NO:234)；
其中，目标序列2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2的抑制效果可反应脱靶效应的程度。即，抑制效果越高，待测缀合物越可能发生脱靶。测得的IC 50值总结于表5中。
表5 siRNA缀合物在psi-CHECK系统中的IC 50
缀合物编号 IC 50/(MOS-5) IC 50/(MOS-5)/IC 50/(GSCM)
缀合物1 1.23nM 37.6
缀合物2 1.98nM 71.2
缀合物3 0.666nM 24.0
缀合物4 0.592nM 29.3
参比缀合物1 0.971nM 20.9
其中，表5中，IC 50/(MOS-5)/IC 50/(GSCM)能够反映不同的siRNA缀合物在达到相同的目标序列抑制活性时所能实现的防脱靶效应。
由表5的结果可知，本公开的siRNA缀合物均显示出与不包含稳定化修饰核苷酸的参比缀合物1相比更大的IC 50/(MOS-5)/IC 50/(GSCM)值，尤其是缀合物2，IC 50/(MOS-5)/IC 50/(GSCM)值是参比缀合物1的3.4倍。因此，在实现相同的目标序列抑制活性时，本公开的siRNA缀合物1-4具有显著降低的脱靶效应。
实验例3 siRNA缀合物在体外psi-CHECK系统中的抑制活性
按照实验例1的方法，测试了缀合物5、缀合物7和参比缀合物2在体外psi-CHECK系统中对目标序列的抑制活性。区别仅在于，对于缀合物5、缀合物7和参比缀合物2分别使用如下所示的目标序列3代替目标序列1，IC 50值总结于表6中：
目标序列3：5’-GGCTTTCAGCTATATGGAT-3’(SEQ ID NO:235)。
表6 siRNA缀合物在psi-CHECK系统中的IC 50
缀合物编号 IC 50/(GSCM)
缀合物5 0.0839nM
缀合物7 0.0253nM
参比缀合物2 0.0488nM
缀合物5、缀合物7和参比缀合物2之间的区别在于：按照5'末端到3'末端的方向，缀合物5正义链的第18个核苷酸上的碱基为C，反义链的第三个核苷酸上具有2'-O-甲氧基乙基修饰基团；缀合物7正义链的第18个核苷酸上的碱基为C，反义链的第五个核苷酸上具有2'-O-甲氧基乙基修饰基团；参比缀合物2正义链的第18个核苷酸上的碱基为A，反义链上没有2'-O-甲氧基乙基修饰基团。
由上述表6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，IC 50在0.01-0.1nM之间。同时，本公开的siRNA缀合物与不包含稳定化修饰核苷酸的参比缀合物2相比具有相当的目标序列抑制活性，其中缀合物7甚至具有比参比缀合物2更高的目标序列抑制活性。
实验例4siRNA缀合物在体外psi-CHECK系统中的抑制活性
按照实验例1的方法，测试了缀合物5、缀合物6、缀合物7、缀合物8和参比缀合物2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5-8和参比缀合物2使用如下所示的目标序列4代替目标序列1：
目标序列4：5’-GGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTTCCGGTTAGGGACTGTATATGGATTT-3’(SEQ ID NO:236)；
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，待测缀合物越可能发生脱靶。测得的IC 50值总结于表7中。
表7 siRNA缀合物在psi-CHECK系统中的IC 50
缀合物编号 IC 50/(MOS-5)
缀合物5 --
缀合物7 --
参比缀合物2 14.5nM
由表7的结果可知，在siRNA浓度为40nM时，并且将与待测缀合物中siRNA反义链部分互补的序列扩增5倍的情况下，仍检测不到siRNA缀合物的脱靶。因此，该实验例说明，本发明提供的siRNA和缀合物，能够显著降低siRNA缀合物的脱靶效应。
实验例5本公开的缀合物在体外(in vitro)的抑制活性
本实验例考察了缀合物1-8、参比缀合物1-2和参比缀合物NC在小鼠肝原代细胞中对HBV基因的抑制活性。
具体步骤如下：
[1]小鼠肝原代细胞分离：
自C57BL/6j-TgN(AlblHBV)44Bri小鼠(以简称44Bri小鼠)体中提取分离出小鼠肝原代细胞。向12孔培养板的每孔中加入1×105细胞/孔的小鼠肝原代细胞液，将细胞板在37℃、5％CO 2培养箱中进行过夜培养。
[2]自由摄取
向每孔中加入1000μL细胞维持培养基(CM Culture medium，北京瑞德百奥生物科技有限公司)。
对于每一待测siRNA缀合物，用1×PBS配制成浓度为20μM(以缀合物中siRNA的量计)的缀合物储液。然后再进行稀释，得到浓度为4μM的缀合物工作液。
在上述每一含有小鼠肝原代细胞的培养孔中加入2.5μL的上述缀合物工作液，得到每个细胞孔中siRNA缀合物终浓度约为10nM(以siRNA的量计)的测试组，每一测试缀合物平行加入2个培养孔。
在另外2个培养孔中，加入2.5μL的参比缀合物工作液，得到每个细胞孔中siRNA缀合物终浓度约为10nM(以siRNA的量计)的阴性对照组。
在另外2个培养孔中，加入2.5μL的PBS溶液，得到空白对照组。
在37℃下，将12孔板置于CO 2培养箱继续培养24小时。
[3]检测
使用离心柱式总RNA抽提试剂盒(购自生工生物工程(上海)股份有限公司，货号：B511361-0100)，根据说明书记载的方法分别提取各孔细胞中的总RNA。
对于每孔细胞，分别取1μg总RNA，使用反转录试剂盒Goldenstar TM RT6 cDNA Synthesis Kit(购自Promega，货号A3500)提供的试剂，其中选取 Goldenstar TM Oligo(dT) 15作为引物，按Reverse Transcription System试剂盒说明书中对各孔细胞的总RNA进行反转录。反应结束后，向反转录反应体系中加入DEPC水80μL，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用
Figure PCTCN2022140698-appb-000058
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HBV和内参基因GAPDH的PCR引物序列如表8所示，每条引物的终浓度为10μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火30s，72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因HBV和内参基因GAPDH的产物W1。产物W1随即依次经过95℃15s，60℃1min，梯度升温至95℃并每0.3℃采集一次荧光信号，95℃15s，实时荧光定量PCR仪分别收集产物W1中目标基因和内参基因GAPDH的溶解曲线，得到目标基因HBV和内参基因GAPDH的Ct值。
表8引物信息
Figure PCTCN2022140698-appb-000059
采用比较Ct(ΔΔCt)法，对各测试组中目标基因HBV mRNA的表达水平进行相对定量计算，计算方法如下：
ΔCt(测试组)＝Ct(测试组目标基因)–Ct(测试组内参基因)
ΔCt(对照组)＝Ct(对照组目标基因)–Ct(对照组内参基因)
ΔΔCt(测试组)＝ΔCt(测试组)-ΔCt(对照组平均)
ΔΔCt(对照组)＝ΔCt(对照组)-ΔCt(对照组平均)
其中，ΔCt(对照组平均)是对照组两个培养孔各自的ΔCt(对照组)的算术平均值。从而，测试组和对照组的每一培养孔均对应一个ΔΔCt值。
以对照组平均值为基准，对测试组HBV mRNA的表达水平进行归一化，定义对照组HBV mRNA表达水平平均值为100％，
测试组HBV mRNA相对表达水平＝2 -ΔΔCt(测试组)×100％
测试组HBV mRNA抑制率＝(1-测试组HBV mRNA相对表达水平)×100％
实验结果如图1所示，图1是显示了10nM浓度的条件下的本公开的缀合物和参比缀合物在小鼠肝原代细胞中的目标序列抑制活性的柱状图。结果表明，本公开的缀合物1-4、缀合物5和缀合物7在10nM浓度时，都具有较好的体 外抑制活性，特别是缀合物2和缀合物4，抑制活性高达99.4％。与不包含稳定化修饰核苷酸的参比缀合物1和参比缀合物2相比，包含稳定化修饰核苷酸的缀合物表现出基本相同甚至更高的活性。
实验例6 siRNA缀合物在小鼠体内(in vivo)对HBV mRNA的抑制
使用乙型肝炎病毒表面抗原诊断试剂盒(酶联免疫法)(上海科华生物)按照说明书记载的方法检测44Bri小鼠血清HbsAg含量，选取S/COV>10的小鼠，随机分组(均为雄性)，每组5只小鼠，分别编号，以皮下注射的方式，向每只小鼠以1mg/kg小鼠体重的剂量(以siRNA计)给予缀合物10或参比缀合物3，siRNA缀合物以含0.2mg/ml(以siRNA计)的siRNA缀合物的1×PBS溶液形式提供，给药体积均为5ml/kg；向另外2组小鼠中的每一只给予1×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天处死动物，分别收集每只小鼠的肝脏组织，用RNA later(Sigma Aldrich公司)保存；向每份肝组织中加入1mL Trizol(Sigma公司)，在Tissuelyset II型全自动组织匀浆仪中破碎3次，每次30s，获得肝组织匀浆，向其中加入0.2mL氯仿，静置3min。在4℃下以12000rpm离心10min，取0.4mL上清。向上清中加入0.5mL异丙醇，室温下静置10min。4℃下以12000rpm离心10min，弃去上清。向沉淀中加入1mL乙醇洗涤沉淀，4℃下以12000rpm离心5min，弃去上清。沉淀中加入70μL DEPC化水，得到提取的总RNA溶液。
对于每一小鼠的肝脏组织总RNA，分别取包含1μg总RNA的总RNA水溶液10.5μL，使用反转录试剂盒Reverse Transcription System(购自Promega公司，货号A3500)，按试剂盒说明书中反转录操作步骤配制为反转录反应体系20μL，对总RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于42℃孵育30min，然后于95℃孵育5min，最后于4℃孵育5min，反应结束后，向反转录反应体系中加入DEPC水80μL，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用SYBR select Master Mix试剂盒(Applied biosystem公司)提供的试剂配制qPCR反应体系20μL，其中，用于扩增目标基因HBV X和内参基因GAPDH的PCR引物序列如下表9所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火30s，72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因HBV X和内参基因GAPDH的产物W。产物W随即依次经过95℃1min，55℃30s，95℃30s的孵育，实时荧光定量PCR仪分别收集产物W中目标基因HBV X和内参基因GAPDH的溶解曲线，得到目标基因HBV X和内参基因GAPDH的 Ct值。
表9引物序列信息
Figure PCTCN2022140698-appb-000060
采用比较Ct(ΔΔCt)法，对各测试组中目标基因HBV mRNA的表达水平进行相对定量计算，计算方法如上述实验例5所述。
图2为给予了1mg/kg(以siRNA计)缀合物10或参比缀合物3以及PBS后，44Bri小鼠肝内HBV mRNA相对表达水平的散点图。图中PBS表示空白对照组。进一步地，各siRNA缀合物对HBV mRNA的抑制率总结于表10中。
表10小鼠体内HBV mRNA的抑制
Figure PCTCN2022140698-appb-000061
缀合物10和参比缀合物3之间的区别在于：按照5'末端到3'末端的方向，缀合物10反义链的第五个核苷酸上具有2'-O-甲氧基乙基修饰基团；参比缀合物3反义链上没有2'-O-甲氧基乙基修饰基团。
由图2和表10可知，本公开的siRNA缀合物在小鼠体内显示出优异的HBV mRNA抑制效果，在1mg/kg的剂量下，缀合物10在小鼠体内的HBV mRNA抑制率可高达84.25％，显示出与不包括稳定化修饰核苷酸的参比缀合物3相近的HBV mRNA抑制活性。
实验例7 siRNA缀合物在小鼠中的毒性效果
将缀合物9、缀合物10、参比缀合物3和参比缀合物4分别用PBS溶解为3mg/ml的溶液(以siRNA缀合物计)。将ICR小鼠(雌雄各半，18-22g重，5-6周龄，购自于斯贝福公司)随机分组，每组6只小鼠(雌雄各半)，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，给药体积均为10mL/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10mL/kg，作为空白对照组。
以给药时间点作为第1天计算，在第15天对测试组和空白对照组的每只小鼠进行眼眶采血，采血量为0.6mL，采血后在37℃下孵育60min后，在4℃下，3000rpm转速离心15min获得血清。进一步使用PM1P000/3全自动血清生化仪(SABA，意大利)检测血清中丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的浓度。结果参见图3A、图3B。图中，PBS表示空白对 照组。
图3A和图3B分别是给予30mg/kg的缀合物9、缀合物10、参比缀合物3、参比缀合物4或PBS后，小鼠血清中ALT和AST浓度的散点图。由图3A和图3B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3后，血清ALT和AST浓度有所升高；而给予本公开的siRNA缀合物后，血清ALT和AST浓度与空白对照组水平相当，表明本公开的siRNA缀合物具有低的肝毒性。另一方面，给予仅在反义链5'-3'端方向第7位包含稳定化修饰核苷酸的参比缀合物4后，小鼠血清ALT和AST的浓度显著升高，表明该参比缀合物可能产生更高的肝毒性。
进一步地，在第15天采血后，处死小鼠并进行剖检，在10％中性缓冲福尔马林固定液中保存并制作病理切片。对病理切片中，肝脂肪变性的严重程度进行评价分级，并作相对比较。
病理切片结果显示，与空白对照相比，给予不包含稳定化修饰核苷酸的参比缀合物3的小鼠中，4只小鼠表现出重度肝细胞变性，具体表现为组织广泛可见肝细胞胞质疏松，较多肝细胞气球样变性，细胞肿胀，胞质呈空泡状，1只小鼠表现出中度肝细胞变性，具体表现为大量肝细胞胞质疏松淡染，部分肝细胞伴有空泡变性，胞质内可见微小的圆形空泡，局部少量肝细胞坏死，胞核固缩或碎裂，1只小鼠表现出轻度肝细胞变性，具体表现为较多肝细胞胞质疏松淡染，显示出比空白对照组更为严重的肝脂肪变性。
与空白对照相比，给予仅在反义链5'-3'端方向第7位包含稳定化修饰核苷酸的参比缀合物4的小鼠中，2只表现出重度肝脂肪变性，4只表现出了中度肝脂肪变性，也显示出比空白对照组的小鼠中严重的肝脂肪变性。
给予本公开的siRNA缀合物9的小鼠中，2只表现出中度肝脂肪变性，3只表现出轻度肝脂肪变性，未见重度或以上的肝细胞变性。给予本公开的siRNA缀合物10的小鼠中，3只表现出中度肝脂肪变性，2只表现出轻度肝脂肪变性，未见重度或以上的肝细胞变性。相比参比缀合物，给予本公开的缀合物的小鼠表现出更低的肝脂肪变性程度。
上述结果表明，与参比缀合物相比，本公开的siRNA缀合物能够有效降低由于脱靶效应导致的肝毒性反应，因此在制备用于HBV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安全性，具有优异的开发前景。
实验例8siRNA缀合物在小鼠原代肝细胞中的靶mRNA抑制活性
HBV转基因小鼠44Bri/J购自北京大学医学部实验动物科学部，于实验前选择S/COV>10的小鼠进行试验。
从44Bri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1×10 5 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得到的小鼠肝原代细胞悬液，将小鼠肝原代细胞接种到培养孔中。加入小鼠肝原代细胞悬液的体积为1mL/孔，小鼠肝原代细胞数量为1×10 5细胞/孔。
用DEPC化水将下面的siRNA缀合物中的每一个分别配制成4μM(以siRNA计)的siRNA缀合物工作液，所用siRNA缀合物分别为缀合物11、缀合物12或参比缀合物5。将参比缀合物NC配制成4μM的参比siRNA NC工作液。
在不同上述加入小鼠肝原代细胞悬液培养孔中，分别加入每个缀合物的siRNA缀合物工作液或参比缀合物NC工作液，均匀混合，加入量为2.5μL/孔，每一siRNA缀合物或参比缀合物NC分别转染3个培养孔，得到含siRNA(以siRNA计，终浓度为10nM)的转染混合物，记为测试组。将另外3个培养孔中的小鼠肝原代细胞悬液记为空白对照组。
将每一含siRNA的转染混合物和空白对照组置于含5％CO 2的培养箱中，在37℃下继续培养24h。
随后，使用TRIZOL(购自SIGMA公司，货号T9424)根据说明书记载的方法提取各孔细胞中的总RNA，得到总RNA水溶液。
对于每孔细胞，分别取包含1μg总RNA的总RNA水溶液，使用反转录试剂盒Goldenstar TM RT6 cDNA Synthesis Kit(购自北京擎科新业生物技术有限公司，货号TSK301M)提供的试剂，其中选取Goldenstar TM Oligo(dT) 17作为引物，按试剂盒说明书中反转录操作步骤配置反转录反应体系20μL，对各孔细胞的总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5min，反应结束后，向反转录反应体系中加入DEPC水80μL，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用
Figure PCTCN2022140698-appb-000062
SYBR qPCR SuperMix Plus试剂盒(购自近岸蛋白质科技有限公司，货号E096-01B)提供的试剂配制qPCR反应体系15μL，其中，用于扩增目标基因HBV X和内参基因GAPDH的PCR引物序列如表9所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火25s，72℃延伸25s，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HBV X和内参基因GAPDH的产物W。产物W随即依次经过95℃1min，55℃30s，95℃30s的孵育，实时荧光定量PCR仪分别收集产物W中目标基因HBV X和内参基因GAPDH的溶解曲线，得到目标基因HBV X和内参基因GAPDH的Ct值。
按照实验例6记载的方法，由Ct值计算自由摄取各siRNA缀合物后，小鼠原代肝细胞中HBV mRNA的相对表达水平和抑制率。结果如图4所示。
图4为分别自由摄取了缀合物11、缀合物12或参比缀合物5以及参比缀合物NC后，44Bri小鼠原代肝细胞中HBV mRNA相对表达水平的柱状图。进一步地，各siRNA缀合物或参比缀合物NC对HBV mRNA的抑制率总结于表11中。
表11小鼠原代肝细胞中HBV mRNA的抑制
Figure PCTCN2022140698-appb-000063
缀合物11、缀合物12和参比缀合物5之间的区别在于：按照5'末端到3'末端的方向，缀合物11反义链的第三个核苷酸上具有2'-O-甲氧基乙基修饰基团；缀合物12反义链的第五个核苷酸上具有2'-O-甲氧基乙基修饰基团；参比缀合物5反义链上没有2'-O-甲氧基乙基修饰基团。
由图4和表11的结果可见，本公开的siRNA缀合物在44Bri小鼠原代肝细胞中显示出优异的HBV mRNA抑制活性，在10nM的siRNA浓度下，HBV mRNA抑制率至少91.77％；特别是，缀合物11在小鼠原代肝细胞中的HBV mRNA抑制率可达93.06％，显示出与对应不包括稳定化修饰核苷酸的参比缀合物5基本相当的HBV mRNA抑制活性。
实验例9 siRNA缀合物在小鼠体内(in vivo)对HBV mRNA的抑制
使用乙型肝炎病毒表面抗原诊断试剂盒(酶联免疫法)(上海科华生物)按照说明书记载的方法检测44Bri小鼠血清HbsAg含量，选取S/COV>10的小鼠，随机分组(均为雄性)，每组5只小鼠，分别编号，以皮下注射的方式，向每只小鼠以1mg/kg小鼠体重的剂量(以siRNA计)给予缀合物11、缀合物2或参比缀合物5，siRNA缀合物以含0.2mg/ml(以siRNA计)的siRNA缀合物的1×PBS溶液形式提供，给药体积均为5ml/kg；向另外2组小鼠中的每一只给予1×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天处死动物，分别收集每只小鼠的肝脏组织，用RNA later(Sigma Aldrich公司)保存；向每份肝组织中加入1mL Trizol(Sigma公司)，在Tissuelyset II型全自动组织匀浆仪中破碎3次，每次30s，获得肝组织匀浆，向其中加入0.2mL氯仿，静置3min。在4℃下以12000rpm离心10min，取0.4mL上清。向上清中加入0.5mL异丙醇，室温下静置10min。4℃下以12000rpm离心10min，弃去上清。向沉淀中加入1mL乙醇洗涤沉淀，4℃下以12000rpm离心5min，弃去上清。沉淀中加入70μL DEPC化水，得到提取的总RNA溶液。
对于每一小鼠的肝脏组织总RNA，分别取包含1μg总RNA的总RNA水溶液10.5μL，使用反转录试剂盒Reverse Transcription System(购自Promega公司，货号A3500)，按试剂盒说明书中反转录操作步骤配制为反转录反应体系20μL，对总RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于42℃孵育30min，然后于95℃孵育5min，最后于4℃孵育5min，反应结束后，向反转录反应体系中加入DEPC水80μL，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用SYBR select Master Mix试剂盒(Applied biosystem公司)提供的试剂配制qPCR反应体系20μL，其中，用于扩增目标基因HBV X和内参基因GAPDH的PCR引物序列如表9所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火30s，72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因HBV X和内参基因GAPDH的产物W。产物W随即依次经过95℃1min，55℃30s，95℃30s的孵育，实时荧光定量PCR仪分别收集产物W中目标基因HBV X和内参基因GAPDH的溶解曲线，得到目标基因HBV X和内参基因GAPDH的Ct值。
按照实验例6记载的方法，由Ct值计算给予各siRNA缀合物后，小鼠体内肝组织中HBV mRNA的相对表达水平和抑制率，结果如图5所示。
图5为给予了1mg/kg(以siRNA计)缀合物11、缀合物12或参比缀合物5以及PBS后，44Bri小鼠肝内HBV mRNA相对表达水平的散点图。图5中，PBS表示空白对照组。进一步地，各siRNA缀合物对HBV mRNA的抑制率总结于表12中。
表12小鼠体内HBV mRNA的抑制
Figure PCTCN2022140698-appb-000064
由图5和表12可知，本公开的siRNA缀合物在小鼠体内显示出优异的HBV mRNA抑制效果，在1mg/kg的剂量下，HBV mRNA抑制率可高达96.31％，并且显示出与对应不包括稳定化修饰核苷酸的参比缀合物5相当的HBV mRNA抑制活性。
实验例10 siRNA缀合物在大鼠中的毒性效果
将缀合物11、缀合物12、参比缀合物5和参比缀合物8分别用PBS溶解 为6mg/ml的溶液(以siRNA缀合物计)。将SD大鼠(均为雄性，0.22-0.28kg重，5-7周龄，购自于维通利华公司)随机分组，每组5只大鼠，分别编号。以颈背部皮下注射的方式，向每只大鼠分别给予上述siRNA缀合物溶液，给药体积均为5mL/kg，作为测试组；另外向一组大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15天处死测试组和空白对照组的每只大鼠并进行剖检，对肝脏进行称重并基于空白对照组进行归一化，在10％中性缓冲福尔马林固定液中保存并制作病理切片，大体剖检和肝重的结果参见表13。对病理切片中，肝脂肪变性和炎症的严重程度进行评价分级并作相对比较。
表13 siRNA缀合物在大鼠中毒性效果
Figure PCTCN2022140698-appb-000065
表13中，％及前面的数字代表相应指标与参比空白对照组的百分比的差值。“↑”代表增加。例如，↑5.43％指的是肝重参比空白对照组增加了5.43％。
缀合物11、缀合物12、参比缀合物5和参比缀合物8之间的区别在于：按照5'末端到3'末端的方向，缀合物11反义链的第三个核苷酸上具有2'-O-甲氧基乙基修饰基团；缀合物12反义链的第五个核苷酸上具有2'-O-甲氧基乙基修饰基团；参比缀合物5反义链上没有2'-O-甲氧基乙基修饰基团；参比缀合物8反义链的第七个和第十二个核苷酸上均具有2'-O-甲氧基乙基修饰基团。
由表13结果可知，给予参比缀合物5和参比缀合物8的大鼠分别增加高达81.31％和84.03％，显示出明显的肝重增加。进一步地，并在大体剖检中显示出较为明显的肝变黄、肿大。而给予缀合物11和缀合物12的大鼠肝重仅分别增加5.45％和3.45％，在大体剖检中与空白对照组相比未见明显异常。
病理切片结果显示，给予参比缀合物5的大鼠中有3例出现了重度以上的肝脂肪变性和轻度或中度的肝脏炎症，具体表现为组织中肝细胞广泛重度脂肪变性，胞质内可见数量不等、大小不等的圆形空泡，少量肝细胞重度肿胀，胞质淡染，静脉周围可见少量炎性细胞浸润。给予参比缀合物8的所有大鼠均显示出极重度的肝脂肪变性和轻度或中度的肝脏炎症，具体表现为组织中肝细胞广泛重度脂肪变性，胞质内可见数量不等、大小不等的圆形空泡，小叶内可见几处炎性细胞小灶性浸润或门静脉周围偶见炎性细胞浸润，少量肝细胞重度肿胀，胞质淡染。
而给予缀合物11的大鼠中，有4只仅发现轻度的脂肪变性，具体表现为肝细胞排列紧密，界限清晰，少量肝细胞轻度脂肪变性，胞质内可见微小圆形空泡，另外1只未发现脂肪变性，且全部大鼠中均未发现明显肝脏炎症；给予缀合物12的大鼠中，2只大鼠的脂肪变性程度为轻度，具体表现为肝细胞排列紧密，界限清晰，部分肝细胞轻度脂肪变性，胞质内可见圆形空泡，3只大鼠的脂肪变性程度为中度，具体表现为肝细胞排列紧密，界限清晰，较多肝细胞轻度脂肪变性，胞质内可见圆形空泡，且全部大鼠中均未发现明显肝脏炎症。
上述结果表明，与参比缀合物相比，本公开的siRNA缀合物能够有效降低由于脱靶效应导致的肝毒性反应，因此在制备用于HBV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安全性，具有优异的开发前景。
实验例11 siRNA缀合物在体外sicheck系统中的脱靶序列抑制活性
按照实验例1记载的方法，对siRNA缀合物在体外sicheck系统中的脱靶序列抑制活性进行了测定，区别仅在于，以缀合物11、缀合物12、缀合物13、缀合物14、缀合物15、缀合物16、参比缀合物5或参比缀合物9代替所测试的siRNA进行测定；并且，所采用的目标序列为如下所示的目标序列5：GGCCGCATTGAAGTTACTGATCCTTCCAAATTGAAGTTACTGATCCTTCCAAATTGAAGTTACTGATCCTTCCAAATTGAAGTTACTGATCCTTCCAAATTGAAGTTACTGATCCTTC(SEQ ID NO:245)
该目标序列5中有多段序列与待测siRNA缀合物中的siRNA反义链部分互补，因此各siRNA缀合物对目标序列3的抑制效果可反映脱靶效应的程度。即，抑制效果越高，该siRNA缀合物越可能发生脱靶。
由剂量-效应曲线和对应的函数，确定当Y＝50％时对应的X 50值，计算获得各siRNA缀合物的脱靶IC 50值＝10^X 50(nM)。.
其结果，参比缀合物5的脱靶IC 50值为218.085pM，参比缀合物9的脱靶IC 50值为202.581pM，即，这两个参比siRNA缀合物在该脱靶IC 50以上的浓度时发生显著脱靶；与此相比，缀合物15的脱靶IC 50值为555.240pM，显著高于参比siRNA缀合物；特别是，其余各siRNA缀合物在全部测试浓度范围内的Renilla的相对残留活性始终高于50％，其中，缀合物11、缀合物12、缀合物13、缀合物14和缀合物16的Renilla的相对残留活性最低依次为55.65％、71.42％、71.38％、65.47％、67.84％，即，上述siRNA缀合物均未发生脱靶。可见，与不包含稳定化修饰核苷酸的参比缀合物5、参比缀合物9相比，包含稳定化修饰核苷酸的各siRNA缀合物均显示出显著更低的脱靶效应。
实验例12 siRNA缀合物在小鼠原代肝细胞中的靶mRNA抑制活性
按照实验例9记载的方法，对siRNA缀合物在小鼠原代肝细胞中的靶mRNA抑制活性进行了测定，区别仅在于，以缀合物11、缀合物12、缀合物 15、缀合物16、参比缀合物5、参比缀合物9、参比缀合物6、参比缀合物7或参比缀合物NC代替所测试的siRNA缀合物进行测定，每一缀合物、参比缀合物或参比缀合物NC在2个培养孔中进行实验。结果如图6所示。
图6为分别自由摄取了缀合物11、缀合物12、缀合物15、缀合物16、参比缀合物5、参比缀合物9、参比缀合物6、参比缀合物7或参比缀合物NC后，44Bri小鼠原代肝细胞中HBV mRNA相对表达水平的柱状图。进一步地，各siRNA缀合物或参比缀合物NC对HBV mRNA的抑制率总结于表14中。
表14小鼠原代肝细胞中HBV mRNA的抑制
Figure PCTCN2022140698-appb-000066
由图6和表14的结果可见，本公开的siRNA缀合物11、12、15和16在44Bri小鼠原代肝细胞中显示出优异的HBV mRNA抑制活性，在10nM的siRNA浓度下，HBV mRNA抑制率至少为78.47％，最高可达85.97％，HBV mRNA抑制活性与参比缀合物5活性相当，并且明显高于参比缀合物9、6和7。其中，参比缀合物5中对应位置为非稳定化修饰的核苷酸，参比缀合物9中对应位置是未修饰的核苷酸，参比缀合物6和7在反义链5'-3'端方向第3-9位之外还包含稳定化修饰核苷酸。
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 (56)

  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中的每一个核苷酸独立地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HBV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为所述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为C 2-C 6烷基、取代的C 2-C 6烷基、C 6-C 8芳基、取代的C 6-C 8芳基中的一种，每个R'独立地为H、C 1-C 6烷基、取代的C 1-C 6烷基、C 6-C 8芳基、取代的C 6-C 8芳基 中的一种，所述取代的C 2-C 6烷基、取代的C 6-C 8芳基或取代的C 1-C 6烷基是指C 2-C 6烷基、C 6-C 8芳基或C 1-C 6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：C 1-C 3烷基、C 6-C 8芳基、C 1-C 3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'-CUAGGAGGCUGUAGGCAUZ 1-3'(SEQ ID NO:1)；
    5'-Z 2AUGCCUACAGCCUCCUAG-3'(SEQ ID NO:2),
    其中，所述Z 1为A,Z 2为U，所述核苷酸序列I中包含位置对应于Z 1的核苷酸Z 3，所述核苷酸序列II中包含位置对应于Z 2的核苷酸Z 4，所述Z 4是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:46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:47所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GGCUUUCAGUUAUAUGGAZ 5-3'(SEQ ID NO:46)；
    5'-Z 6UCCAUAUAACUGAAAGCC-3'(SEQ ID NO:47),
    其中Z 5为U,Z 6为A，所述核苷酸序列I中包含位置对应于Z 5的核苷酸Z 7，所述核苷酸序列II中包含位置对应于Z 6的核苷酸Z 8，所述Z 8是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:91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:92所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-CCUUGAGGCAUACUUCAAZ 9-3'(SEQ ID NO:91)；
    5'-Z 10UUGAAGUAUGCCUCAAGG-3'(SEQ ID NO:92),
    其中Z 9为A,Z 10为U，所述核苷酸序列I中包含位置对应于Z 9的核苷酸Z 11，所述核苷酸序列II中包含位置对应于Z 10的核苷酸Z 12，所述Z 12是所述反义链5'末端的第一个核苷酸；
    或者，所述核苷酸序列I与SEQ ID NO:136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:137所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-UGCUAUGCCUCAUCUUCUZ 13-3'(SEQ ID NO:136)；
    5'-Z 14AGAAGAUGAGGCAUAGCA-3'(SEQ ID NO:137),
    其中Z 13为A,Z 14为U，所述核苷酸序列I中包含位置对应于Z 13的核苷酸Z 15，所述核苷酸序列II中包含位置对应于Z 14的核苷酸Z 16，所述Z 16是所述反义链5'末端的第一个核苷酸。
  12. 如权利要求11所述的siRNA，其中，所述第一段核苷酸序列是如SEQ ID NO:1所示的核苷酸序列；或者，所述第一段核苷酸序列是如SEQ ID NO:46所示的核苷酸序列；或者，所述第一段核苷酸序列是如SEQ ID NO:91所示的核苷酸序列；或者，所述第一段核苷酸序列是如SEQ ID NO:136所示的核苷酸序列。
  13. 如权利要求11或12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2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO:46所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:47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:91所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:92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:136所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:137所示的核苷酸序列之间不多于1个核苷酸差异。
  14. 如权利要求13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Z 4位置处的差异，且Z 4选自A、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:47所示的核苷酸序列之间的差异包括Z 8位置处的差异，且Z 8选自U、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:92所示的核苷酸序列之间的差异包括Z 12位置处的差异，且Z 12选自A、G或C；
    或者，所述核苷酸序列II与SEQ ID NO:137所示的核苷酸序列之间的差异包括Z 16位置处的差异，且Z 16选自A、G或C。
  15. 如权利要求1-14中任意一项所述的siRNA，其中，所述Z 3是与Z 4互 补的核苷酸；或者所述Z 7是与Z 8互补的核苷酸；或者所述Z 11是与Z 12互补的核苷酸；或者所述Z 15是与Z 16互补的核苷酸。
  16. 如权利要求1-15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。
  17. 如权利要求16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。
  18. 如权利要求17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。
  19. 如权利要求1-18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，所述反义链的长度为19-26个核苷酸；并且所述核苷酸序列I是SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:7所示的核苷酸序列：
    5'-CUAGGAGGCUGUAGGCAUZ 3-3'(SEQ ID NO:3)；
    5'-CUAGGAGGCUGUAGGCACZ 3-3'(SEQ ID NO:4)；
    5'-CUAGGAGGCUGUAGGCAGZ 3-3'(SEQ ID NO:5)；
    5'-CUAGGAGGCUGUAGGCAAZ 3-3'(SEQ ID NO:6)；
    5'-Z 4AUGCCUACAGCCUCCUAG-3'(SEQ ID NO:7),
    其中，Z 3选自A、U、G或C，Z 4是与Z 3互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:52所示的核苷酸序列：
    5'-GGCUUUCAGUUAUAUGGAZ 7-3'(SEQ ID NO:48)；
    5'-GGCUUUCAGUUAUAUGGCZ 7-3'(SEQ ID NO:49)；
    5'-GGCUUUCAGUUAUAUGGGZ 7-3'(SEQ ID NO:50)；
    5'-GGCUUUCAGUUAUAUGGUZ 7-3'(SEQ ID NO:51)；
    5'-Z 8UCCAUAUAACUGAAAGCC-3'(SEQ ID NO:52),
    其中，Z 7选自A、U、G或C，Z 8是与Z 7互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:97所示的核苷酸序列：
    5'-CCUUGAGGCAUACUUCAAZ 11-3'(SEQ ID NO:93)；
    5'-CCUUGAGGCAUACUUCAUZ 11-3'(SEQ ID NO:94)；
    5'-CCUUGAGGCAUACUUCAGZ 11-3'(SEQ ID NO:95)；
    5'-CCUUGAGGCAUACUUCACZ 11-3'(SEQ ID NO:96)；
    5'-Z 12UUGAAGUAUGCCUCAAGG-3'(SEQ ID NO:97),
    其中，Z 11选自A、U、G或C，Z 12是与Z 11互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140或SEQ ID NO:141所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:142所示的核苷酸序列：
    5'-UGCUAUGCCUCAUCUUCUZ 15-3'(SEQ ID NO:138)；
    5'-UGCUAUGCCUCAUCUUCAZ 15-3'(SEQ ID NO:139)；
    5'-UGCUAUGCCUCAUCUUCGZ 15-3'(SEQ ID NO:140)；
    5'-UGCUAUGCCUCAUCUUCCZ 15-3'(SEQ ID NO:141)；
    5'-Z 16AGAAGAUGAGGCAUAGCA-3'(SEQ ID NO:142),
    其中，Z 15选自A、U、G或C，Z 16是与Z 15互补的核苷酸。
  20. 如权利要求19所述的siRNA，其中Z 3为A，Z 4为U；或者Z 7为U，Z 8为A；或者Z 11为A，Z 12为U；或者Z 15为A，Z 16为U。
  21. 如权利要求1-20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2'-氟代修饰的核苷酸。
  22. 如权利要求21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2'-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。
  23. 如权利要求1-22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2'-氟代修饰的核苷酸。
  24. 如权利要求23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2'-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。
  25. 如权利要求1-24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的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，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，所述第二段核苷酸序列是指HBV基因表达的mRNA中与第一段核苷酸序列相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
  26. 如权利要求25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GUA，所述核苷酸序列IV的碱基组成为UAC；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UGUA，所述核苷酸序列IV的碱基组成为UACA。
  27. 如权利要求25所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:46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UU，所述核苷酸序列IV的碱基组成为AA；或者，所述核苷酸序列III和IV的长度均为3个核苷酸，所述核苷酸序列III的碱基组成为UUU，所述核苷酸序列IV的碱基组成为AAA；或者，所述核苷酸序列III和IV的长度均为4个核苷酸，所述核苷酸III的碱基组成为GUUU，所述核苷酸序列IV的碱基组成为AAAC。
  28. 如权利要求25所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:91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GA，所述核苷酸序列IV的碱基组成为UC；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CGA，所述核苷酸序列IV的碱基组成为UCG；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CCGA，所述核苷酸序列IV的碱基组成为UCGG。
  29. 如权利要求25所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:136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C，核苷酸序列IV的碱基为G；或者，所述核苷酸序列III和IV的长度均为2个核苷酸，所述核苷酸序列III的碱基组成为GC，所述核苷酸序列IV的碱基组成为GC；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UGC，所述核苷酸序列IV的碱基组成为GCA；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CUGC，所述核苷酸序列IV的碱基组成为GCAG。
  30. 如权利要求1-29中任意一项所述的siRNA，其中所述siRNA还含有核苷酸序列V，所述核苷酸序列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。
  31. 如权利要求30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，所述第三段核苷酸序列是指HBV基因表达的mRNA中与第一段核苷酸序列或第二段核苷酸序列相邻、并且长度与所述核苷酸序列V相等的核苷酸序列。
  32. 如权利要求1-31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、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，所述反义链含有如SEQ ID NO:8所示的核苷酸序列：
    5'-CUAGGAGGCUGUAGGCAUZ 3-3'(SEQ ID NO:3)；
    5'-CUAGGAGGCUGUAGGCACZ 3-3'(SEQ ID NO:4)；
    5'-CUAGGAGGCUGUAGGCAGZ 3-3'(SEQ ID NO:5)；
    5'-CUAGGAGGCUGUAGGCAAZ 3-3'(SEQ ID NO:6)；
    5'-Z 4AUGCCUACAGCCUCCUAGUA-3'(SEQ ID NO:8),
    其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C， 并且Z 4是与Z 3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:13所示的核苷酸序列：
    5'-UACUAGGAGGCUGUAGGCAUZ 3-3'(SEQ ID NO:9)；
    5'-UACUAGGAGGCUGUAGGCACZ 3-3'(SEQ ID NO:10)；
    5'-UACUAGGAGGCUGUAGGCAGZ 3-3'(SEQ ID NO:11)；
    5'-UACUAGGAGGCUGUAGGCAAZ 3-3'(SEQ ID NO:12)；
    5'-Z 4AUGCCUACAGCCUCCUAGUACA-3'(SEQ ID NO:13),
    其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C，并且Z 4是与Z 3互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:53所示的核苷酸序列：
    5'-GGCUUUCAGUUAUAUGGAZ 7-3'(SEQ ID NO:48)；
    5'-GGCUUUCAGUUAUAUGGCZ 7-3'(SEQ ID NO:49)；
    5'-GGCUUUCAGUUAUAUGGGZ 7-3'(SEQ ID NO:50)；
    5'-GGCUUUCAGUUAUAUGGUZ 7-3'(SEQ ID NO:51)；
    5'-Z 8UCCAUAUAACUGAAAGCCAA-3'(SEQ ID NO:53),
    其中，所述Z 8是反义链5'末端的第一个核苷酸，Z 7选自A、U、G或C，并且Z 8是与Z 7互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或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'-UUGGCUUUCAGUUAUAUGGAZ 7-3'(SEQ ID NO:54)；
    5'-UUGGCUUUCAGUUAUAUGGCZ 7-3'(SEQ ID NO:55)；
    5'-UUGGCUUUCAGUUAUAUGGGZ 7-3'(SEQ ID NO:56)；
    5'-UUGGCUUUCAGUUAUAUGGUZ 7-3'(SEQ ID NO:57)；
    5'-Z 8UCCAUAUAACUGAAAGCCAAAC-3'(SEQ ID NO:58),
    其中，所述Z 8是反义链5'末端的第一个核苷酸，Z 7选自A、U、G或C，并且Z 8是与Z 7互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:98所示的核苷酸序列：
    5'-CCUUGAGGCAUACUUCAAZ 11-3'(SEQ ID NO:93)；
    5'-CCUUGAGGCAUACUUCAUZ 11-3'(SEQ ID NO:94)；
    5'-CCUUGAGGCAUACUUCAGZ 11-3'(SEQ ID NO:95)；
    5'-CCUUGAGGCAUACUUCACZ 11-3'(SEQ ID NO:96)；
    5'-Z 12UUGAAGUAUGCCUCAAGGUC-3'(SEQ ID NO:98),
    其中，所述Z 12是反义链5'末端的第一个核苷酸，Z 11选自A、U、G或C，并且Z 12是与Z 11互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101或SEQ ID NO:102所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:103所示的核苷酸序列：
    5'-GACCUUGAGGCAUACUUCAAZ 11-3'(SEQ ID NO:99)；
    5'-GACCUUGAGGCAUACUUCAUZ 11-3'(SEQ ID NO:100)；
    5'-GACCUUGAGGCAUACUUCAGZ 11-3'(SEQ ID NO:101)；
    5'-GACCUUGAGGCAUACUUCACZ 11-3'(SEQ ID NO:102)；
    5'-Z 12UUGAAGUAUGCCUCAAGGUCGG-3'(SEQ ID NO:103),
    其中，所述Z 12是反义链5'末端的第一个核苷酸，Z 11选自A、U、G或C，并且Z 12是与Z 11互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140或SEQ ID NO:141所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:143所示的核苷酸序列：
    5'-UGCUAUGCCUCAUCUUCUZ 15-3'(SEQ ID NO:138)；
    5'-UGCUAUGCCUCAUCUUCAZ 15-3'(SEQ ID NO:139)；
    5'-UGCUAUGCCUCAUCUUCGZ 15-3'(SEQ ID NO:140)；
    5'-UGCUAUGCCUCAUCUUCCZ 15-3'(SEQ ID NO:141)；
    5'-Z 16AGAAGAUGAGGCAUAGCAGC-3'(SEQ ID NO:143),
    其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 15选自A、U、G或C，并且Z 16是与Z 15互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146或SEQ ID NO:147所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:148所示的核苷酸序列：
    5'-GCUGCUAUGCCUCAUCUUCUZ 15-3'(SEQ ID NO:144)；
    5'-GCUGCUAUGCCUCAUCUUCAZ 15-3'(SEQ ID NO:145)；
    5'-GCUGCUAUGCCUCAUCUUCGZ 15-3'(SEQ ID NO:146)；
    5'-GCUGCUAUGCCUCAUCUUCCZ 15-3'(SEQ ID NO:147)；
    5'-Z 16AGAAGAUGAGGCAUAGCAGCAG-3'(SEQ ID NO:148),
    其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 15选自A、U、G或C，并且Z 16是与Z 15互补的核苷酸。
  33. 如权利要求1-32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。
  34. 如权利要求1-33中任意一项所述的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。
  35. 如权利要求1-34中任意一项所述的siRNA，其中，所述siRNA为siHBVa1-M1、siHBVa1-M2、siHBVa2-M1、siHBVa2-M2、siHBVa3-M1、siHBVa3-M2、siHBVa4-M1、siHBVa4-M2、siHBVa5-M1、siHBVa5-M2、siHBVa6-M1、siHBVa6-M2、siHBVa7-M1、siHBVa7-M2、siHBVa8-M1、siHBVa8-M2、siHBVb1-M1、siHBVb1-M2、siHBVb2-M1、siHBVb2-M2、siHBVb3-M1、siHBVb3-M2、siHBVb4-M1、siHBVb4-M2、siHBVb5-M1、siHBVb5-M2、siHBVb6-M1、siHBVb6-M2、siHBVb7-M1、siHBVb7-M2、siHBVb8-M1、siHBVb8-M2、siHBVc1-M1、siHBVc1-M2、siHBVc2-M1、siHBVc2-M2、siHBVc3-M1、siHBVc3-M2、siHBVc4-M1、siHBVc4-M2、siHBVc5-M1、siHBVc5-M2、siHBVc6-M1、siHBVc6-M2、siHBVc7-M1、siHBVc7-M2、siHBVc8-M1、siHBVc8-M2、siHBVd1-M1、siHBVd1-M2、siHBVd2-M1、siHBVd2-M2、siHBVd3-M1、siHBVd3-M2、siHBVd4-M1、siHBVd4-M2、siHBVd5-M1、siHBVd5-M2、siHBVd6-M1、siHBVd6-M2、siHBVd7-M1、siHBVd7-M2、siHBVd8-M1或siHBVd8-M2中的一种。
  36. 如权利要求1-35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。
  37. 如权利要求36所述的siRNA，其中，所述siRNA为siHBVa1-M1S、siHBVa1-M2S、siHBVa2-M1S、siHBVa2-M2S、siHBVa3-M1S、siHBVa3-M2S、siHBVa4-M1S、siHBVa4-M2S、siHBVa5-M1S、siHBVa5-M2S、siHBVa6-M1S、siHBVa6-M2S、siHBVa7-M1S、siHBVa7-M2S、siHBVa8-M1S、siHBVa8-M2S、siHBVb1-M1S、siHBVb1-M2S、siHBVb2-M1S、siHBVb2-M2S、siHBVb3-M1S、 siHBVb3-M2S、siHBVb4-M1S、siHBVb4-M2S、siHBVb5-M1S、siHBVb5-M2S、siHBVb6-M1S、siHBVb6-M2S、siHBVb7-M1S、siHBVb7-M2S、siHBVb8-M1S、siHBVb8-M2S、siHBVc1-M1S、siHBVc1-M2S、siHBVc2-M1S、siHBVc2-M2S、siHBVc3-M1S、siHBVc3-M2S、siHBVc4-M1S、siHBVc4-M2S、siHBVc5-M1S、siHBVc5-M2S、siHBVc6-M1S、siHBVc6-M2S、siHBVc7-M1S、siHBVc7-M2S、siHBVc8-M1S、siHBVc8-M2S、siHBVd1-M1S、siHBVd1-M2S、siHBVd2-M1S、siHBVd2-M2S、siHBVd3-M1S、siHBVd3-M2S、siHBVd4-M1S、siHBVd4-M2S、siHBVd5-M1S、siHBVd5-M2S、siHBVd6-M1S、siHBVd6-M2S、siHBVd7-M1S、siHBVd7-M2S、siHBVd8-M1S或siHBVd8-M2S中的一种。
  38. 如权利要求1-37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。
  39. 如权利要求38所述的siRNA，其中，所述siRNA为siHBVa1-M1P1、siHBVa1-M2P1、siHBVa2-M1P1、siHBVa2-M2P1、siHBVa3-M1P1、siHBVa3-M2P1、siHBVa4-M1P1、siHBVa4-M2P1、siHBVa5-M1P1、siHBVa5-M2P1、siHBVa6-M1P1、siHBVa6-M2P1、siHBVa7-M1P1、siHBVa7-M2P1、siHBVa8-M1P1、siHBVa8-M2P1、siHBVa1-M1SP1、siHBVa1-M2SP1、siHBVa2-M1SP1、siHBVa2-M2SP1、siHBVa3-M1SP1、siHBVa3-M2SP1、siHBVa4-M1SP1、siHBVa4-M2SP1、siHBVa5-M1SP1、siHBVa5-M2SP1、siHBVa6-M1SP1、siHBVa6-M2SP1、siHBVa7-M1SP1、siHBVa7-M2SP1、siHBVa8-M1SP1、siHBVa8-M2SP1、siHBVb1-M1P1、siHBVb1-M2P1、siHBVb2-M1P1、siHBVb2-M2P1、siHBVb3-M1P1、siHBVb3-M2P1、siHBVb4-M1P1、siHBVb4-M2P1、siHBVb5-M1P1、siHBVb5-M2P1、siHBVb6-M1P1、siHBVb6-M2P1、siHBVb7-M1P1、siHBVb7-M2P1、siHBVb8-M1P1、siHBVb8-M2P1、siHBVb1-M1SP1、siHBVb1-M2SP1、siHBVb2-M1SP1、siHBVb2-M2SP1、siHBVb3-M1SP1、siHBVb3-M2SP1、siHBVb4-M1SP1、siHBVb4-M2SP1、siHBVb5-M1SP1、siHBVb5-M2SP1、siHBVb6-M1SP1、siHBVb6-M2SP1、siHBVb7-M1SP1、siHBVb7-M2SP1、siHBVb8-M1SP1、siHBVb8-M2SP1、siHBVc1-M1P1、siHBVc1-M2P1、siHBVc2-M1P1、siHBVc2-M2P1、siHBVc3-M1P1、siHBVc3-M2P1、siHBVc4-M1P1、siHBVc4-M2P1、siHBVc5-M1P1、siHBVc5-M2P1、siHBVc6-M1P1、siHBVc6-M2P1、siHBVc7-M1P1、siHBVc7-M2P1、siHBVc8-M1P1、siHBVc8-M2P1、siHBVc1-M1SP1、siHBVc1-M2SP1、siHBVc2-M1SP1、siHBVc2-M2SP1、siHBVc3-M1SP1、siHBVc3-M2SP1、siHBVc4-M1SP1、siHBVc4-M2SP1、siHBVc5-M1SP1、siHBVc5-M2SP1、siHBVc6-M1SP1、siHBVc6-M2SP1、siHBVc7-M1SP1、siHBVc7-M2SP1、siHBVc8-M1SP1、siHBVc8-M2SP1、siHBVd1-M1P1、siHBVd1-M2P1、siHBVd2-M1P1、siHBVd2-M2P1、siHBVd3-M1P1、siHBVd3-M2P1、siHBVd4-M1P1、siHBVd4-M2P1、siHBVd5-M1P1、 siHBVd5-M2P1、siHBVd6-M1P1、siHBVd6-M2P1、siHBVd7-M1P1、siHBVd7-M2P1、siHBVd8-M1P1、siHBVd8-M2P1、siHBVd1-M1SP1、siHBVd1-M2SP1、siHBVd2-M1SP1、siHBVd2-M2SP1、siHBVd3-M1SP1、siHBVd3-M2SP1、siHBVd4-M1SP1、siHBVd4-M2SP1、siHBVd5-M1SP1、siHBVd5-M2SP1、siHBVd6-M1SP1、siHBVd6-M2SP1、siHBVd7-M1SP1、siHBVd7-M2SP1、siHBVd8-M1SP1或siHBVd8-M2SP1中的一种。
  40. 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中的每一个核苷酸独立地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HBV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2'-O-甲氧基乙基修饰的核苷酸。
  41. 如权利要求40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2'-O-甲氧基乙基修饰的核苷酸。
  42. 如权利要求40或41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2'-O-甲氧基乙基修饰的核苷酸。
  43. 如权利要求40-42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II的第2、6、14、16个核苷酸，如果不是2'-O-甲氧基乙基修饰的核苷酸的话，为2'-氟代修饰的核苷酸。
  44. 如权利要求43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2'-O-甲氧基乙基修饰的核苷酸的话，为2'-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。
  45. 如权利要求40-44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的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，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，所述第二段核苷酸序列是指HBV基因表达的mRNA中与第一段核苷酸序列相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
  46. 如权利要求40-45中任意一项所述的siRNA，其中所述siRNA还含有核苷酸序列V，所述核苷酸序列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2'-O-甲氧基乙基修饰的核苷酸，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。
  47. 一种siRNA缀合物，所述siRNA缀合物含有权利要求1-46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连接或非共价连接，每个所述靶向基团选自能够和细胞表面受体结合的配体。
  48. 如权利要求47所述的siRNA缀合物，其中，所述siRNA缀合物为具有式(403)所示的结构缀合物或其水可溶性盐，其中Nu是siHBVa1-M1S、siHBVa2-M1S、siHBVa3-M1S、siHBVa4-M1S、siHBVb1-M1S、siHBVb2-M1S、siHBVb3-M1S、siHBVb4-M1S、siHBVc1-M1S siHBVc2-M1S、siHBVc3-M1S、siHBVc4-M1S、siHBVd1-M1S、siHBVd2-M1S、siHBVd3-M1S和siHBVd4-M1S中的一种或多种所示的siRNA形成的siRNA基团。
  49. 如权利要求48所述的siRNA缀合物，其中，所述Nu中的每个所述稳定化修饰核苷酸均为2'-O-甲氧基乙基修饰的核苷酸。
  50. 一种药物组合物，所述药物组合物含有如权利要求1-46中任意一项所述的siRNA，和/或如权利要求47-49中任意一项所述的siRNA缀合物，以及药学上可接受的载体。
  51. 权利要求1-46中任意一项所述的siRNA，和/或权利要求47-49中任意一项所述的siRNA缀合物和/或权利要求50所述的药物组合物在制备用于治疗和/或预防与HBV基因表达的mRNA水平相关的疾病或者症状的药物中的用途。
  52. 如权利要求51所述的用途，其中所述与HBV基因表达的mRNA水平相关的疾病或症状是慢性肝病、炎症、纤维化疾病和增生性疾病中的至少一种。
  53. 一种治疗和/或预防与HBV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有效量的权利要求1-46中任意一项所述的siRNA，和/或权利要求47-49中任意一项所述的siRNA缀合物和/或权利要求50所述的药物组合物。
  54. 如权利要求53所述的方法，其中，所述与HBV基因表达的mRNA水平相关的疾病或症状是慢性肝病、炎症、纤维化疾病和增生性疾病中的至少一种。
  55. 一种抑制细胞中HBV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1-46中任意一项所述的siRNA，和/或权利要求47-49中任意一项所述的siRNA缀合物和/或权利要求50所述的药物组合物与所述细胞接触。
  56. 一种试剂盒，所述试剂盒包含权利要求1-46中任意一项所述的siRNA，和/或权利要求47-49中任意一项所述的siRNA缀合物和/或权利要求50所述的药物组合物。
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CN110997919A (zh) * 2017-12-01 2020-04-10 苏州瑞博生物技术有限公司 双链寡核苷酸、含双链寡核苷酸的组合物与缀合物及制备方法和用途
CN111050807A (zh) * 2017-12-01 2020-04-21 苏州瑞博生物技术有限公司 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
CN111655849A (zh) * 2018-08-21 2020-09-11 苏州瑞博生物技术有限公司 一种核酸、含有该核酸的药物组合物和缀合物及其用途
CN111655297A (zh) * 2018-09-30 2020-09-11 苏州瑞博生物技术有限公司 一种siRNA缀合物及其制备方法和用途

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