WO2021114227A1 - 一种含胺转染试剂和制备方法及转染复合物 - Google Patents

一种含胺转染试剂和制备方法及转染复合物 Download PDF

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WO2021114227A1
WO2021114227A1 PCT/CN2019/125165 CN2019125165W WO2021114227A1 WO 2021114227 A1 WO2021114227 A1 WO 2021114227A1 CN 2019125165 W CN2019125165 W CN 2019125165W WO 2021114227 A1 WO2021114227 A1 WO 2021114227A1
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transfection
amine
transfection complex
present disclosure
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PCT/CN2019/125165
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English (en)
French (fr)
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张鸿雁
杨志伟
黄金宇
王秀莲
吴林玉
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苏州瑞博生物技术股份有限公司
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Priority to CN201980102539.9A priority Critical patent/CN114761376A/zh
Priority to PCT/CN2019/125165 priority patent/WO2021114227A1/zh
Publication of WO2021114227A1 publication Critical patent/WO2021114227A1/zh

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/30—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and unsaturated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to the field of biomedicine, in particular to an amine-containing transfection reagent that can be used to deliver biologically active agents, a method for preparing the amine-containing transfection reagent, and a transfection complex containing the amine-containing transfection reagent.
  • Liposome is an artificial membrane with a double-layer structure containing lipids, generally containing phospholipids, cholesterol and other components, forming nano-lipid particles in a solution. Liposomes can be used to encapsulate and deliver drugs, and to deliver drugs into cells. Liposomes can generally be used to deliver small molecule drugs, and there are already many liposomal small molecule drugs on the market. In addition, liposomes can also be used to encapsulate nucleic acids, transfect nucleic acids into cells, or deliver them to target tissues and enter cells. In the art, amine-containing lipid compounds (ie, transfection reagents) are commonly used to deliver nucleic acid molecules.
  • Chinese Patent CN 103380113B describes a variety of amine-containing lipid compounds.
  • the amine groups in these lipid compounds have a strong electrostatic binding effect with DNA or RNA, so they can be used for the delivery of nucleic acid molecules.
  • this amine-containing lipid compound sometimes brings certain toxicity. Therefore, how to improve the activity of liposomes in delivering nucleic acid drugs while reducing toxicity is still a problem that needs to be solved in the art.
  • the present disclosure provides an amine-containing transfection reagent.
  • the transfection reagent and the transfection complex containing the transfection reagent have better biological activity and lower toxicity, and are suitable for use in biologically active agents, especially nucleic acid drugs. In vivo delivery.
  • an amine-containing transfection reagent which is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
  • Y 1 is selected from C2-C10 alkylene or substituted C2-C10 alkylene;
  • Each Y 2 is the same or different, and is independently selected from a C2-C6 alkylene group or a substituted C2-C6 alkylene group;
  • Each R 1 is the same or different and is independently selected from H or the group represented by formula (Ii), and each R 2 is independently a group represented by formula (I-ii),
  • each R a and each R b are independently selected from C6-C20 linear alkyl groups, and each R c is independently selected from one of the non-amine hydrophilic groups,
  • the present disclosure also provides a method for preparing an amine-containing transfection reagent.
  • the amine-containing transfection reagent is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the method includes:
  • the compound represented by formula (301) is contacted with the compound represented by formula (701) in the presence of a coupling reagent and coupling reaction conditions, and under conditions sufficient to produce the compound represented by formula (I), Isolate the compound represented by formula (I);
  • the group Y 1, Y 2, R a , R 2 is in the range defined as described above, the Z is a leaving group 701.
  • the present disclosure also provides a transfection complex, which contains a key lipid, and the key lipid is the above-mentioned amine-containing transfection reagent provided by the present disclosure.
  • the present disclosure also provides the use of the transfection complex of the present disclosure in the preparation of drugs for treating and/or preventing pathological conditions or diseases caused by the expression of specific genes in cells.
  • the present disclosure also provides a method for treating and/or preventing a pathological condition or disease caused by the expression of a specific gene, the method comprising administering to a subject suffering from the pathological condition or disease the method of the present disclosure Transfection complex.
  • the present disclosure also provides a method for inhibiting the expression of a specific gene in a cell, the method comprising contacting the cell with the transfection complex of the present disclosure.
  • the present disclosure also provides a kit, which includes the transfection complex provided by the present disclosure.
  • Figure 1 shows the transfection complex prepared with the compound of the present disclosure shown in Example 3 and the comparative transfection complex at the doses of 1 mg/kg and 0.5 mg/kg, the liver tissues of BALB/c mice Inhibition rate of ApoB mRNA expression level.
  • Figure 2 shows the expression of ApoB mRNA in liver tissues of BALB/c mice when the transfection complex prepared with the compound of the present disclosure and the comparative transfection complex shown in Example 3 at a dose of 0.1 mg/kg The level of inhibition rate.
  • Figure 3 shows the effect of the transfection complex prepared with the compound of the present disclosure shown in Example 3 and the comparative transfection complex on the concentration of total cholesterol (CHO) in serum in BALB/c mice.
  • Figure 4 shows the effect of the transfection complex prepared with the compound of the present disclosure shown in Example 3 and the comparative transfection complex on the serum triglyceride (TG) concentration in BALB/c mice.
  • a dash that is not between two letters or between two symbols is used to indicate the position of the point of attachment of the substituent.
  • -C 1 -C 10 alkyl-NH 2 is connected through C 1 -C 10 alkyl.
  • alkoxy refers to an alkyl group with a specified number of carbon atoms connected by 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 ⁇ pentoxy.
  • Alkoxy groups generally have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by oxygen bridges.
  • halogen refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (for example, rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry .
  • the subject refers to a human patient suffering from a specific disease.
  • the present disclosure provides an amine-containing transfection reagent for the delivery of biologically active agents.
  • the amine-containing transfection reagent forms a transfection complex (usually in lipids) with a biologically active agent as a pharmaceutical active ingredient.
  • biologically active agent includes, but is not limited to, one of drug molecules, pharmaceutical compositions, drug complexes, prodrugs that can be delivered through the transfection complex of the present disclosure and has therapeutic and/or prophylactic activity. Multiple, or pharmaceutically acceptable salts of any one or more of them.
  • the bioactive agent includes small molecule drugs, functional oligonucleotides or drug proteins, or their prodrugs or pharmaceutically acceptable salts, especially those described in the present disclosure below.
  • an amine-containing transfection reagent which is a compound of the structure represented by formula (I) or a pharmaceutically acceptable salt thereof:
  • Y 1 is selected from C2-C10 alkylene or substituted C2-C10 alkylene;
  • Each Y 2 is the same or different, and is independently selected from a C2-C6 alkylene group or a substituted C2-C6 alkylene group;
  • Each R 1 is the same or different and is independently selected from H or the group represented by formula (Ii), and each R 2 is independently a group represented by formula (I-ii),
  • each R a and each R b are independently selected from C6-C20 linear alkyl groups, and each R c is independently selected from one of the non-amine hydrophilic groups,
  • Y 1 is a C2-C10 linear alkylene group. In some embodiments, Y 1 is a C3-C5 linear alkylene group. In some embodiments, Y 1 is butylene.
  • alkylene is a linear or branched divalent saturated hydrocarbon group composed of carbon atoms and hydrogen atoms, located between two other chemical groups and used to connect these two other chemical groups.
  • Y 2 is a C2-C6 alkylene group with one hydroxy substituent. In some embodiments, each Y 2 is independently a C2-C3 alkylene group with one hydroxy substituent. In some embodiments, each Y 2 is 2-hydroxypropylene.
  • each R 2 is independently a group shown in (I-ii), wherein each R b is independently selected from a C6-C20 linear alkyl group, and each R Each c is independently selected from one of non-amine hydrophilic groups.
  • R c is selected from non-amine hydrophilic groups.
  • each R c is independently selected from a hydroxyl group, a sulfhydryl group, a carboxyl group, a phosphoric acid group, or a polyethylene glycol group. In some embodiments, each R c is a hydroxyl group. In some embodiments, each R b is independently a C8-C16 linear alkyl group. In some embodiments, all R b are the same, for example, each R b is dodecyl.
  • each R 1 is independently selected from H or the group represented by (Ii). Wherein, two R 1 may both be H, both are groups represented by (Ii), or one R 1 is H and the other R 1 is a group represented by (Ii). In some embodiments, both R 1 are H. In some embodiments, one R 1 is H and the other R 1 is a group represented by (Ii). In conjunction with R c described in the present disclosure, the present disclosure provides The amine-containing transfection reagent showed better results.
  • each R a is independently selected from C6-C20 straight chain alkyl. In some embodiments, each R a is independently selected from linear C10-C18 alkyl group. In some embodiments, R a are all the same, for example, each R a are both pentadecyl.
  • the amine-containing transfection reagent of the present disclosure is selected from one or more of the compounds represented by formulas (101)-(103) and pharmaceutically acceptable salts thereof.
  • the pharmaceutically acceptable salt can be selected from pharmaceutically acceptable salts commonly used in the art, as long as the salt is within a reasonable range of medical evaluation and is suitable for contact with human and animal tissues and organs, and does not show Additional toxicity, irritation, allergic reactions, etc., and are consistent with a reasonable benefit/risk ratio.
  • the pharmaceutically acceptable salt may be a pharmaceutically acceptable acid addition salt formed with an organic acid and/or an inorganic acid, such as acetate, glutamate, lactate, adipate, benzoic acid
  • an organic acid and/or an inorganic acid such as acetate, glutamate, lactate, adipate, benzoic acid
  • an organic acid and/or an inorganic acid such as acetate, glutamate, lactate, adipate, benzoic acid
  • One or more of salt malate, citrate, mandelate, succinate, methanesulfonate, hydrochloride, hydrobromide, sulfate, and phosphate.
  • the preparation of the compound represented by formula (I) includes at least in an organic solvent, in the presence of an organic base and coupling reaction conditions, and under conditions sufficient to produce the compound represented by formula (I) , Contacting the compound represented by formula (301) with the compound represented by formula (701) to isolate the compound represented by formula (I);
  • the group Y 1, Y 2, R a , R 2 is in the range defined as described above, the Z is a leaving group 701.
  • the organic solvent may be one or more of halogenated alkane, ether, nitrile and amide solvents.
  • the organic solvent is dimethylformamide (DMF).
  • the amount of the organic solvent can be 4-30 L/mol, for example, 6-20 L/mol.
  • the organic base may be a tertiary amine organic base, such as triethylamine or N,N-diisopropylethylamine (DIPEA).
  • DIPEA N,N-diisopropylethylamine
  • the molar ratio of the organic base to the compound represented by formula (301) is 1:1-20:1, and in some embodiments, 3:1-10:1.
  • the leaving group Z 701 can be a leaving group commonly used in the art, such as halogen, -OCOR, -OTs, -ONO 2 or -OH.
  • the leaving group Z 701 is halogen.
  • the leaving group Z 701 is bromine (Br).
  • the compound represented by formula (701) can be easily synthesized by those skilled in the art, or can be obtained commercially, for example, when the leaving group Z 701 is bromine, it can be obtained from commercially available methyl 4-bromocrotonate and Starting from fatty alcohols with different carbon chain lengths, various compounds represented by formula (701) can be easily prepared by transesterification methods known in the art.
  • the molar ratio of the compound represented by formula (701) to the compound represented by formula (301) may be 2:1-10:1 , For example, 2:1-6:1.
  • the coupling reaction conditions refer to conditions that are sufficient to cause the leaving group Z 701 to leave and the compound represented by formula (701) reacts with the compound represented by formula (301) to form a covalent connection, and are sufficient to produce formula (I).
  • the condition of the compound shown in) means that the above-mentioned coupling reaction is fully carried out, and the degree is sufficient to make enough of the compound represented by formula (701) react with the compound represented by formula (301) to form a covalent connection, thereby generating formula (I)
  • the reaction of the compound shown can be carried out at a suitable temperature, such as 40-90°C, for 1-15 hours. In some embodiments, the reaction is carried out at 50°C for 2 h.
  • the progress of the reaction can be monitored by thin layer chromatography or HPLC, and the end point of the reaction can be determined by specific indicators such as the content of reactants/products in the reaction mixture.
  • Any suitable separation method can be used to separate the compound represented by formula (I) from the reaction mixture.
  • the obtained reaction product may be a mixture of compounds that all conform to the structure represented by formula (I) but have different amounts of R 1. Therefore, in some embodiments, the reaction product can be washed and dried, and then eluted with an eluent gradient on column chromatography, and the eluent fractions with a single compound composition can be collected and concentrated to separate a single product.
  • the eluent fractions to be combined can be determined by real-time detection of whether the collected eluent has a single composition by, for example, thin layer chromatography (TLC), HPLC or LC-MS.
  • the eluent can be, for example, a mixed solution of dichloromethane (DCM) and methanol (MeOH), and the volume ratio of dichloromethane to methanol is 50:1-10:1.
  • the method for preparing the compound represented by formula (301) includes contacting the compound represented by formula (302) with a deprotection reagent in an organic solvent under deprotection reaction conditions to isolate the compound represented by formula (301) Show compounds.
  • R 2 , Y 1 , Y 2 are the same as the foregoing, and R 304 is an amino protecting group.
  • the organic solvent may be one or more of halogenated alkanes, ethers, alcohols and amide solvents; in some embodiments, the organic solvent is ethanol or methanol. Relative to the compound represented by formula (302), the amount of the organic solvent can be 4-30 L/mol, for example, 10-25 L/mol.
  • the amino protecting group R 304 can be various amino protecting groups known to those skilled in the art, such as tert-butoxycarbonyl (BOC), benzyloxy (CBz), benzyl (Bn), trimethylsilyl Group, trifluoroacetyl (CF 3 CO) or acetyl group.
  • two R 304 groups on the same nitrogen atom together form a single divalent protecting group, such as a phthaloyl group.
  • the deprotection reagent and its amount can be determined according to the amino protecting group R 304 .
  • the amino protecting group is a phthaloyl group.
  • the deprotection reagent can be, for example, hydrazine hydrate, and the molar ratio of the deprotection reagent to the compound represented by formula (302) can be 4:1-20: 1. Such as 5:1-8:1.
  • the reaction can be carried out at a suitable temperature, such as 40-90°C, for 1-15 hours. In some embodiments, the reaction is carried out under solvent reflux conditions for 2h.
  • the progress of the reaction can be monitored by chromatography or chromatography-mass spectrometry. Any suitable separation method can be used to separate the compound represented by formula (301) from the reaction product.
  • the reaction product after the reaction product is washed and dried, the compound represented by formula (301) produced by the reaction can be separated using, for example, column chromatography.
  • the reaction product can be directly used in the subsequent reaction without further processing.
  • Y 2 is 2-hydroxy-C2-C6 alkylene
  • the preparation method of the compound represented by formula (302) includes in the presence of an organic solvent and an organic base. Under the conditions of the addition ring-opening reaction, the compound represented by formula (303) is contacted with the compound represented by formula (703) to isolate the compound represented by formula (302).
  • R 2 , Y 1 , and R 304 are the same as the foregoing, and Y 703 is a covalent bond or a C1-C4 alkylene group.
  • the organic solvent may be one or more of halogenated alkanes, ethers, alcohols, and amide solvents; in some embodiments, the organic solvent is DMF. Relative to the compound represented by formula (303), the amount of the organic solvent can be 4-20 L/mol, for example, 7-15 L/mol.
  • the oxirane group in the compound represented by formula (703) is ring-opened to form a 2-hydroxyethylene group connected to the amino group, thereby forming the compound represented by formula (302) together with the Y 703 group The Y 2 group in.
  • the molar ratio of the compound represented by formula (703) to the compound represented by formula (303) is 2:1-4:1, for example, it may be 2.6:1-3.2:1.
  • the organic base is a tertiary amine, such as triethylamine or N,N,-diisopropylethylamine.
  • the molar ratio of the organic base to the compound represented by formula (303) is 1:1-10:1, and in some embodiments, it is 1.2:1-5:1.
  • the reaction can be carried out at an appropriate temperature, such as 90-150°C, for 5-15 hours. In some embodiments, the reaction is carried out at 110-130°C for 6-12 h.
  • any suitable separation method can be used to separate the compound represented by formula (302) from the reaction product.
  • the compound represented by formula (302) produced by the reaction can be separated using, for example, column chromatography.
  • the reaction product can be directly used in the subsequent reaction without further processing.
  • each R c is a hydroxyl group
  • the preparation method of the compound represented by formula (303) includes adding the diamine represented by formula (304) in an organic solvent under the conditions of the addition ring-opening reaction.
  • the compound is contacted with the ethylene oxide derivative represented by the formula (704) to isolate the compound represented by the formula (303).
  • Y 1 and R b are the same as the foregoing.
  • the organic solvent may be one or more of halogenated alkanes, ethers, alcohols, and amide solvents; in some embodiments, the organic solvent is ethanol. Relative to the compound represented by formula (304), the amount of the organic solvent can be 3-20 L/mol, for example, 4-10 L/mol.
  • the oxirane group in the compound represented by formula (704) is ring-opened to form a 2-hydroxyethylene group connected to the amino group, thereby forming the compound represented by formula (303) together with the R b group The R 2 group in.
  • the molar ratio of the compound represented by formula (704) to the compound represented by formula (304) is 2:1-4:1, for example, it may be 2.1:1-2.6:1.
  • the reaction can be carried out at an appropriate temperature, such as 25-70°C, for 2-10 hours. In some embodiments, the reaction is carried out at 50°C for 4-6 h. Any suitable separation method can be used to separate the compound represented by formula (303) from the reaction product. In some embodiments, the compound represented by formula (303) can be obtained after filtering, washing, and drying. In some embodiments, the reaction product can be directly used in the subsequent reaction without further processing.
  • the compound represented by the above formula (703), the compound represented by the formula (704), and the compound represented by the formula (304) can be easily prepared by those skilled in the art according to the existing published literature, or obtained commercially.
  • the compound represented by formula (304) is 1,4-butanediamine, which is easily commercially available;
  • Y 703 is a methylene group, and two R 304 on the same nitrogen atom
  • the compound represented by formula (703) is commercially available N-(2,3-epoxypropyl)phthalamide; when R b is dodecyl
  • the compound represented by formula (704) is 1,2-epoxytetradecane that is easily commercially available.
  • other raw materials used in the present disclosure are also commercially available raw materials or raw materials that can be easily prepared according to the existing disclosure.
  • the present disclosure provides a transfection complex comprising the amine-containing transfection reagent of the present disclosure.
  • transfection complex generally refers to a composition used to deliver a biologically active agent (such as a nucleic acid, a drug protein, or a small molecule) to a cell or tissue in vivo or in vitro.
  • a biologically active agent such as a nucleic acid, a drug protein, or a small molecule
  • the transfection complex without biologically active agent can be produced and sold separately as a product. Therefore, the transfection complex provided in the present disclosure may contain key lipids, which refer to the key lipids described in the present disclosure.
  • the amine-containing transfection reagent may contain key lipids, which refer to the key lipids described in the present disclosure.
  • the transfection complex further comprises helper lipids and/or pegylated lipids.
  • the molar ratio between the key lipid, auxiliary lipid and PEGylated lipid can be varied within a relatively wide range. In some embodiments, the molar ratio is (15-100):(0-85):(0-50); in some embodiments, the molar ratio is (19.7-80):(19.7-80): (0.3-50); Alternatively, the molar ratio can be (50-70):(20-40):(3-30).
  • auxiliary lipid used in the present disclosure generally refers to other auxiliary lipids suitable for preparing and forming transfection complexes in addition to the amine-containing transfection reagent provided in the present disclosure, that is, the key lipid.
  • Suitable auxiliary lipids can be auxiliary lipids commonly used in the art.
  • the auxiliary lipids are selected from, but not limited to, cholesterol, cholesterol analogs, cholesterol derivatives, sterols (including phytosterols, animal sterols, and hopanes). Class), or any one or more of neutral or cationic lipids that are known to achieve or promote the introduction of exogenous bioactive agents into cells or tissues.
  • auxiliary lipid may be used in the formulation of the transfection complexes described in this disclosure.
  • exemplary, but non-limiting, neutral or cationic lipids that can be used to prepare the transfection complexes provided in the present disclosure can be one or more lipids selected from the group consisting of: BMOP(N-(2- (Bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propylammonium bromide), DDPES (dipalmitoylphosphatidylethanolamine 5-carboxyspermine amide) ), DSPC, CTAB: DOPE (a preparation of cetyl trimethylammonium bromide (CATB) and DOPE), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), DOPE (Dioleylphosphatidylethanolamine), DMG, DMAP (4-dimethylaminopyridine
  • the neutral lipid can also be selected from the following commercially available cationic lipid mixtures, for example, (N, NI, NII, NIII-tetramethyl-N, NI, NII, NIII-tetrapalmitylsperamine (TMTPS) and dioleoylphosphatidylethanolamine (DOPE) 1:1.5 (M/M) preparation) , GS2888 with LIPOFECTAMINE LIPOFECTAMINE TFXN TM , TRANSFAST TM , vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine (also known as double C14-amidine), And others.
  • TTPS NIII-tetramethyl-N, NI, NII, NIII-tetrapalmitylsperamine
  • DOPE dioleoylphosphatidylethanolamine
  • auxiliary lipids Any combination or mixture of the auxiliary lipids listed above may also be contemplated for use in the transfection complexes of the present disclosure.
  • the following patent documents, patent applications or references are incorporated into the present disclosure by reference in their entirety, particularly their disclosures on transfection agents containing cations and mediums that can be used to form the transfection complex of the present disclosure.
  • Sex auxiliary lipids U.S. Patents 6,075,012, 6,020,202, 5,578,475, 5,736,392, 6,051,429, 6,376,248, 5,334,761, 5,316,948, 5,674,908, 5,834,439, 6,110,916, 6,399,663, 6,716,000882, 5,627,159; PCT/US/2004430633, Publication Nos. US/9926825, publication number is WO0027795A1; PCT/US/04016406, publication number is WO04105697; and PCT/US2006/019356, publication number is WO07130073
  • pegylated lipid generally refers to a lipid that is covalently conjugated to one or more polyethylene glycol moieties.
  • the PEGylated lipids used in the transfection complex embodiments of the present disclosure include: phosphatidylethanolamine (PE)-based PEGylated lipids, for example, 1,2-dimyristoyl-sn-glycerol- Ethanolamine 3-phosphate-N-[methoxy(polyethylene glycol)-MW], where MW represents the average molar mass of the polyethylene glycol moiety.
  • PE phosphatidylethanolamine
  • Such dimyristoyl-PEG-PE lipids are collectively referred to as 14:0PEG(MW)PE.
  • the average MW of the polyethylene glycol portion can be, for example, 25, 350, 550, 750, 1000, 2000, 3000, 5000, 6000, 8000, or 12000.
  • the fatty acid chains of phosphatidylethanolamine-based PEGylated lipids may include, for example, 1,2-dioleoyl (such as in the case of 18:1 PEG(MW)PE), 1,2-dipalmitoyl (such as in the case of 16:0PEG(MW)PE) or 1,2-distearoyl (such as for 18:0PEG(MW)PE).
  • PEGylated lipids based on phosphatidylethanolamine include, for example, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[MOD(polyethylene glycol)-MW] , Also known as DSPE-MODPEG (MW), where MOD represents a functional moiety, such as an amine, biotin, carboxylic acid, folate, maleimide, PDP or carboxyfluorescein moiety.
  • the average molar mass MW of the polyethylene glycol moiety can be, for example, 2000 or 5000.
  • the pegylated lipids used in the embodiments described in the present disclosure also include ceramide-based pegylated lipids, for example, N-octanoyl-sphingosine-1- ⁇ succinyl[methoxy( Polyethylene glycol)MW] ⁇ , also known as C8PEG(MW)ceramide, where the MW is, for example, 750, 2000, or 5000.
  • the fatty acid moiety may have an N-palmitoyl (C16) group (such as in the case of C16PEG(MW) ceramide).
  • the PEGylated lipid used in the transfection complexes of the present disclosure is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene Diol)-2000].
  • the transfection complexes provided in the present disclosure are usually colloidal preparations, but may also be dried preparations.
  • the transfection complex provided in the present disclosure is an aggregate, which can be unilamellar or multilamellar liposomes, vesicles, micelles, and amorphous aggregates.
  • the transfection complex of the present disclosure further includes a bioactive agent.
  • biologically active agent can be a composition, complex, compound, or molecule that has a biological effect, or modifies, causes, promotes, enhances, blocks, or reduces biological effects, or enhances or restricts biological effects.
  • the second molecule may, but does not have to be an endogenous molecule (e.g., a molecule normally found in the target cell, such as a protein or nucleic acid).
  • Biological effects can be, but are not limited to: effects that stimulate or cause an immune response; effects that affect biological processes in cells, tissues, or organisms (for example, animals); effects that affect biological processes in pathogens or parasites Effect; an effect that produces or causes a detectable signal; an effect that modulates the expression of a protein or polypeptide; an effect that terminates or inhibits the expression of a protein or polypeptide; or an effect that causes or enhances the expression of a protein or polypeptide.
  • a suitable bioactive agent may include a molecule that is capable of forming a transfection complex with the amine-containing transfection reagent described in the present disclosure, and when delivered to the interior of one or more cells or When delivered to tissues in vivo or in vitro, it will cause a biological response.
  • the bioactive agent used in the embodiments described in this disclosure may be a cationic, neutral or anionic agent.
  • the bioactive agent is a small molecule, a functional oligonucleotide or a drug protein, or a prodrug or a pharmaceutically acceptable salt of any of them.
  • prodrug is also referred to as “prodrug” or “prodrug”, which refers to the chemical structure modification of the drug, which is inactive or less active in vitro, and is enzymatically modified in vivo. Or non-enzymatic conversion to release active drugs and compounds that exert pharmacological effects.
  • the biologically active agent can be selected from, but not limited to: nucleic acids, polypeptides, antibodies, oligopeptides, therapeutic peptides or protein molecules, peptide nucleic acids (PNA), cationic, anionic or neutral organic Molecules or drugs, or their pharmaceutically acceptable salts.
  • nucleic acids polypeptides, antibodies, oligopeptides, therapeutic peptides or protein molecules, peptide nucleic acids (PNA), cationic, anionic or neutral organic Molecules or drugs, or their pharmaceutically acceptable salts.
  • PNA peptide nucleic acids
  • the bioactive agent is a functional oligonucleotide, or a prodrug or a pharmaceutically acceptable salt thereof.
  • the functional oligonucleotide can generate stable and specific hybridization with the target sequence, using RNA activation (RNAa), RNA interference (RNAi), antisense nucleic acid technology or exon
  • RNAa RNA activation
  • RNAi RNA interference
  • the principle of exon skipping technology is to up-regulate or down-regulate the expression of target genes, or lead to alternative splicing of mRNA.
  • the functional oligonucleotide can also be a nucleic acid structure that produces stable and specific binding with the target protein, such as an aptamer.
  • polynucleotides such as mRNA itself or fragments thereof
  • amine-containing transfection reagents provided in the present disclosure to achieve transfection delivery to cells.
  • the concept of "functional oligonucleotide” can also encompass mRNA or fragments thereof.
  • the functional oligonucleotide can interact with the target sequence, thereby affecting the normal function of the target sequence molecule, such as causing mRNA fragmentation or translation repression or exon skipping to trigger mRNA alternative splicing.
  • the functional oligonucleotide may be substantially complementary, substantially reverse complementary, or completely reverse complementary to the bases of the target sequence.
  • the functional oligonucleotide may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of the target sequence.
  • the functional oligonucleotide may contain 1, 2, or 3 bases that are not complementary to the target sequence.
  • the functional oligonucleotide is selected from deoxyribonucleic acid or ribonucleic acid, and modified deoxyribonucleic acid or ribonucleic acid.
  • the functional oligonucleotide is single-stranded or double-stranded.
  • the functional oligonucleotide may be single-stranded DNA, RNA, or DNA-RNA chimera, or double-stranded DNA, RNA, or DNA-RNA hybrids (hybrids).
  • functional oligonucleotides suitable for inclusion in the transfection complex of the present disclosure may be small interfering RNA (siRNA), microRNA (microRNA), anti-microRNA (antimiR), micro RNA antagonists (antagomir), microRNA mimics (microRNA mimics), decoy oligonucleotides (decoy), immune stimulators (immune stimulatory), G-quadruplex, alternative splices (splice) altering), single-stranded RNA (ssRNA), antisense nucleic acid (antisense), nucleic acid aptamer (Nucleic Acid Aptamer), small activating RNA (small activating RNA, saRNA), stem-loop RNA (stem-loop RNA) or DNAkind of.
  • siRNA small interfering RNA
  • microRNA microRNA
  • anti-microRNA anti-microRNA
  • antiagomir microRNA mimics
  • decoy oligonucleotides decoy
  • immune stimulators i
  • the functional oligonucleotide suitable for inclusion in the transfection complex of the present disclosure may be the oligonucleotide disclosed in WO2009082607A2, WO2009073809A2 or WO2015006740A2, the entire contents of which are incorporated by reference This disclosure.
  • functional oligonucleotides suitable for inclusion in the transfection complexes of the present disclosure are double-stranded oligonucleotides.
  • the functional oligonucleotide suitable for inclusion in the transfection complex of the present disclosure is siRNA.
  • the bioactive agent is an example of a functional oligonucleotide.
  • the transfection complex of the present disclosure can deliver functional oligonucleotides to cells and regulate the expression of specific genes in these cells. These cells may be various cells.
  • the cell is a hepatocyte.
  • the specific gene may be an endogenous gene expressed in liver cells, or a pathogen gene that multiplies in liver cells.
  • the genes expressed in hepatocytes may be, for example, ApoB, ApoC3, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, HCV genes.
  • the gene expressed in hepatocytes is HBV gene, ANGPTL3 gene or APOC3 gene.
  • HBV gene refers to a gene whose sequence is shown in Genbank registration number NC_003977.1
  • ANGPTL3 gene refers to a gene whose mRNA sequence is shown in Genbank registration number NM_014495.3
  • APOC3 gene refers to the gene whose mRNA sequence is shown in Genbank registration number NM_000040.1.
  • the "target sequence” is a target mRNA.
  • target mRNA refers to the mRNA corresponding to a gene that is abnormally expressed in a cell, and it can be either the mRNA corresponding to the over-expressed gene or the mRNA corresponding to the under-expressed gene. Since most diseases result from overexpression of mRNA, in this disclosure, target mRNA especially refers to the mRNA corresponding to the overexpressed gene.
  • the target mRNA may be ApoB, ApoC3, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, HCV genes corresponding to MRNA.
  • the target mRNA may be the mRNA corresponding to the HBV gene, or the mRNA expressed by the ANGPTL3 gene, or the mRNA expressed by the APOC3 gene.
  • the functional oligonucleotide in the transfection complex of the present disclosure is a small interfering RNA (siRNA) is described in detail. This does not mean that the bioactive agent in the transfection complex of the present disclosure can only be siRNA. According to the detailed description of the transfection complex, it is conceivable that other biologically active agents, especially other functional oligonucleotides, and the amine-containing transfection reagent provided in the present disclosure will have similar effects when forming a transfection complex.
  • siRNA small interfering RNA
  • siRNA contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group, and a base.
  • the nucleotide group contains a phosphate group, a ribose group, and a base.
  • functional siRNA is about 12-40 nucleotides in length, and in some embodiments about 15-30 nucleotides, each nucleotide in the siRNA can be independently It is a modified or unmodified nucleotide. In order to increase stability, at least one nucleotide in the siRNA is a modified nucleotide.
  • siRNA described in the following embodiments has higher activity, higher stability, lower off-target effect and/or lower toxicity, and thus can be used as the biological activity in the present disclosure. Agent.
  • each nucleotide in the siRNA in the transfection complex of the present disclosure is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand, wherein the The sense strand includes nucleotide sequence 1, and the antisense strand includes nucleotide sequence 2.
  • nucleotide sequence 1 and nucleotide sequence 2 are equal, and both are 10, 11, 12, and 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides, and at least partially reverse complementary to form a complementary double-stranded region, the nucleotide sequence 2 At least a part is complementary to the first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence in the target mRNA.
  • the length of the nucleotide sequence 1 and the nucleotide sequence 2 are both 19, 20 or 21 nucleotides.
  • the length of the nucleotide sequence 1 and the nucleotide sequence 2 are both 19 nucleotides.
  • the siRNA means that at a dose of 3 mg/kg (calculated as siRNA), it can inhibit at least 50% hepatitis B virus gene expression, at least 50% angiopoietin-like protein 3 gene expression, or at least 50%. % Apolipoprotein C3 gene expression siRNA. In some embodiments, the siRNA is capable of inhibiting at least 55%, 60%, 65%, 70%, 75% or 80% of HBV gene, ANGPTL3 gene or APOC3 gene expression at a dose of 3 mg/kg.
  • the length of the nucleotide sequence 1 and the first nucleotide sequence are the same, and no more than 3 nucleotides difference; the length of the nucleotide sequence 2 and the nucleotide sequence B Equal and no more than 3 nucleotide differences; the nucleotide sequence B is a nucleotide sequence that is completely reverse complementary to the first nucleotide sequence.
  • these specific nucleotide differences will not significantly reduce the target gene suppression ability of the transfection complex, and these transfection complexes containing specific nucleotide differences are also protected by the present disclosure. Within range.
  • the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • basically reverse complementation means that there are no more than 3 base mismatches between the two nucleotide sequences involved; essentially reverse complementarity means that two There is no more than one base mismatch between nucleotide sequences; complete complementarity means that there is no base mismatch between two nucleotide sequences.
  • nucleotide difference between a nucleotide sequence and another nucleotide sequence, which means that the base type of the nucleotide at the same position has changed compared with the latter, for example, in
  • one nucleotide base in the latter is A
  • the corresponding nucleotide base at the same position in the former is U, C, G or T
  • nucleotide difference at the position In some embodiments, when an abasic nucleotide or a nucleotide analog is substituted for the nucleotide at the original position, it can also be considered that there is a nucleotide difference at that position.
  • the nucleotide sequence 1 is not more than one nucleotide difference from the first nucleotide sequence, and/or the nucleotide sequence 2 is different from the nucleotide sequence B does not differ by more than 1 nucleotide.
  • the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B includes in the direction from the 5'end to the 3'end, the first nucleotide sequence on the nucleotide sequence 2 A difference in the Z'position of a nucleotide.
  • the last nucleotide Z on the nucleotide sequence 1 is a nucleotide complementary to Z'.
  • the sense strand further contains a nucleotide sequence 3
  • the antisense strand further contains a nucleotide sequence 4
  • the length of the nucleotide sequence 3 and the nucleotide sequence 4 are equal and Both are 1-4 nucleotides
  • the nucleotide sequence 3 is connected to the 5'end of the nucleotide sequence 1
  • the nucleotide sequence 4 is connected to the 3 of the nucleotide sequence 2.
  • the nucleotide sequence 4 is complementary to the second nucleotide sequence
  • the second nucleotide sequence refers to the target mRNA that is adjacent to the first nucleotide sequence and has the same length as the first nucleotide sequence.
  • the nucleotide sequence 4 is the same as the nucleotide sequence. In some embodiments, the nucleotide sequence 3 and the nucleotide sequence 4 are substantially reverse complementary or completely reverse complementary. Therefore, the length of the sense strand and antisense strand can be 19-23 nucleotides.
  • the siRNA further contains a nucleotide sequence 5, which has a length of 1 to 3 nucleotides, and is connected to the 3'end of the antisense strand, thereby constituting a The 3'overhang of the antisense strand; in some embodiments, the length of the nucleotide sequence 5 is 1 or 2 nucleotides.
  • the ratio of the length of the sense strand and the antisense strand of the siRNA may be 19/20, 19/21, 20/21, 20/22, 21/22, 21/23, 22/ 23, 22/24, 23/24 or 23/25.
  • the length of the nucleotide sequence 5 is 2 nucleotides, and in the direction from the 5'end to the 3'end, the nucleotide sequence 5 is two consecutive deoxythymidine nuclei.
  • Nucleotide, two consecutive uracil nucleotides, or complementary to the third nucleotide sequence the third sequence refers to the target mRNA adjacent to the first nucleotide sequence, or and The second segment of nucleotide sequence is adjacent and has a nucleotide sequence equal in length to nucleotide sequence 5.
  • the ratio of the length of the sense strand and the antisense strand of the siRNA is 19/21 or 21/23. At this time, the siRNA has better target mRNA silencing activity.
  • the nucleotides in the siRNA are each independently a modified or unmodified nucleotide. In some embodiments, the siRNA does not contain modified nucleotide groups; in some embodiments, the siRNA contains modified nucleotide groups.
  • siRNA in the art, there are many ways to modify siRNA in the art, such as backbone modification (also called internucleotide linkage modification, such as phosphate group modification), ribose group modification and base modification (for example, see Watts, JK, GFDeleavey and MJDamha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008.13 (19-20): p.842-55, the entire content of which is incorporated into this disclosure by reference).
  • backbone modification also called internucleotide linkage modification, such as phosphate group modification
  • ribose group modification and base modification for example, see Watts, JK, GFDeleavey and MJDamha, Chemically modified siRNA: tools and applications.
  • Drug Discov Today, 2008.13 (19-20): p.842-55 the entire content of which is incorporated into this disclosure by reference).
  • modified nucleotide refers to a nucleotide whose ribose group is modified, such as a nucleotide or nucleotide analog formed by replacing the 2'hydroxyl group with another group. , Or nucleotides where the base on the nucleotide is a modified base.
  • At least one nucleotide in the sense strand or the antisense strand is a modified nucleotide
  • at least one phosphate group is a phosphate group with a modified group
  • at least a part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain in the sense strand and the antisense strand is a phosphate group with a modified group and/or has The ribose group of the modification group (or the modified phosphate group and/or the modified ribose group).
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
  • the fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (207).
  • Non-fluorinated modified nucleotides refer to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group.
  • each non-fluorinated modified nucleotide is independently selected from among nucleotides or nucleotide analogues formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorinated group.
  • Nucleotides formed by replacing the hydroxyl group at the 2'position of the ribose group with a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-modified nucleotides One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotides are methoxy modified nucleotides (2'-OMe), as shown in formula (208).
  • the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (209).
  • the 2'-amino modified nucleotide (2'-NH 2 ) is represented by formula (210).
  • the 2'-deoxynucleotide (DNA) is represented by formula (211).
  • Nucleotide analogs refer to those that can replace nucleotides in nucleic acids, but whose structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine Group.
  • the nucleotide analogs can be, for example, isonucleotides, bridged nucleic acid (BNA) nucleotides, or acyclic nucleotides.
  • BNA nucleotides refer to nucleotides that are constrained or inaccessible.
  • BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosaccharide condensed bridge structure.
  • the bridge is incorporated into the 2'- and 4'- positions of the ribose ring to provide a 2', 4'-BNA nucleotide, such as LNA, ENA, cET BNA, where LNA is represented by formula (212)
  • ENA is shown in equation (213)
  • cET BNA is shown in equation (214).
  • Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of nucleotides, such as unlocking nucleic acid (UNA) nucleotides or glycerol nucleic acid (GNA) nucleotides, where UNA is represented by formula (215) As shown, GNA is shown in formula (216).
  • UNA unlocking nucleic acid
  • GNA glycerol nucleic acid
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotide refers to a compound formed by changing the position of a base in a nucleotide on the ribose ring, for example, a base moved from the 1'-position of the ribose ring to the 2'-position or 3'-position.
  • the compound is represented by formula (217) or (218).
  • Base represents a base, such as A, U, G, C, or T; R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from one of heteronucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, and the methoxy-modified nucleotide means that the 2'-hydroxyl group of the ribose group is substituted with a methoxy group. And the nucleotides formed.
  • fluoro-modified nucleotides In the above and below, “fluoro-modified nucleotides”, “2'-fluoro-modified nucleotides”, “nucleotides in which the 2'-hydroxyl group of the ribose group is substituted with fluorine” and “2' -Fluorinated ribose group” has the same meaning, and both refer to the compound with the structure shown in formula (207) formed by replacing the 2'-hydroxyl of the nucleotide with fluorine; “Methoxy modified nucleotide”, “2 '-Methoxy modified nucleotides”, “nucleotides in which the 2'-hydroxyl group of a ribose group is substituted by a methoxy group” and “2'-methoxyribosyl” have the same meaning, and they all refer to nucleotides The 2'-hydroxyl group of the ribose group is substituted with a methoxy group to
  • the siRNA is an siRNA with the following modifications: according to the direction from the 5'end to the 3'end, the siRNA is at positions 7, 8, and 9 of the nucleotide sequence 1 in the sense strand of the siRNA.
  • the nucleotides are -fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the nucleotide sequence of the nucleotide sequence 2
  • the nucleotides at positions 2, 6, 14, and 16 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides; in some embodiments,
  • the siRNA is an siRNA with the following modifications: or in the direction from the 5'end to the 3'end, the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence 1 in the sense
  • the nucleotide contains a phosphate group modification.
  • the phosphoric acid group modification in one embodiment is a phosphorthioate modification represented by the following formula (201), that is, a sulfur atom is substituted for the non-bridging oxygen atom in the phosphodiester bond , Thereby replacing the phosphodiester bond with the phosphorothioate bond.
  • the modification can stabilize the structure of the siRNA and maintain the high specificity and affinity of base pairing.
  • the phosphorothioate group linkage exists in at least one of the following positions: the first and the second at either end of the sense strand or the antisense strand Between nucleotides; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above.
  • the phosphorothioate group linkages are present at all the above positions except the 5'end of the sense chain.
  • the phosphorothioate group linkages are present at all of the above-mentioned positions except the 3'end of the sense chain.
  • the phosphorothioate group linkage is present in at least one of the following positions:
  • connection between the second nucleotide and the third nucleotide of the 5'end of the antisense strand
  • connection between the second nucleotide and the third nucleotide of the 3'end of the antisense strand
  • the 5'terminal nucleotide of the antisense strand sequence of the siRNA molecule is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides may have the structure shown in formula (202):
  • nucleotides modified by 5'-phosphate analogues are well-known to those skilled in the art, for example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017,35(3):238-48 disclosed the following 4 kinds of nucleotides as shown in formulas (203)-(206):
  • R represents a group selected from the group consisting of H, OH, F and methoxy
  • Base represents a base selected from A, U, C, G, or T.
  • the nucleotides modified by 5'-phosphate nucleotides or 5'-phosphate analogues are nucleosides containing E-vinylphosphonate (E-VP) represented by formula (203) Acid, a nucleotide containing a 5'-phosphate modification represented by formula (202) or a nucleotide containing a 5'-phosphorothioate modification represented by formula (205).
  • E-VP E-vinylphosphonate
  • Tables 1A-1F show siRNAs that can be used as bioactive agents.
  • the transfection complexes provided in the present disclosure using these siRNAs also have unexpectedly low toxicity characteristics while maintaining high activity.
  • the capital letters C, G, U, A indicate the base composition of nucleotides;
  • the lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a 2'-methoxy modified nucleotide;
  • lowercase The letter f indicates that the adjacent nucleotide to the left of the letter f is a 2'-fluoro-modified nucleotide;
  • the lowercase letter s indicates that the connection between the two adjacent nucleotides to the left and right of the letter s is thio Phosphate group connection;
  • P1 indicates that the nucleotide adjacent to the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, in some embodiments it is a vinyl phosphate modification Of nucleotides, 5'-phosphate modified nucleotides or phosphorothioate modified nucleotides.
  • modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications, and methods for preparing nucleoside monomers with corresponding modifications and The method of introducing modified nucleotide groups into siRNA is also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared by known methods.
  • the nucleoside monomer refers to the RNA sequence to be prepared in the solid phase
  • the "unmodified or modified RNA phosphoramidite" used in the synthesis of phosphoramidite, solid-phase phosphoramidite synthesis is a well-known method for synthesizing RNA in the art.
  • the method usually includes connecting the nucleoside monomers sequentially in a 3'to 5'direction according to the nucleotide type and sequence of the sense strand or the antisense strand in the siRNA to be prepared, Synthesize the sense strand and antisense strand.
  • each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization to obtain the sense strand connected to the solid phase carrier and the one connected to the solid phase carrier.
  • Antisense strand remove the protective group and cut with the solid phase carrier, separate and purify separately, obtain the sense strand or antisense strand of siRNA, and anneal.
  • RNA phosphoramidite also refers to nucleoside phosphoramidites. Unless otherwise specified, the nucleoside monomers used in the present disclosure are all commercially available.
  • Other conditions for the solid-phase synthesis include deprotection conditions of nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, conditions for capping reactions, types and amounts of capping reagents,
  • the oxidation reaction conditions, the type and amount of the oxidizing reagent, the vulcanization reaction conditions, and the type and amount of the vulcanizing reagent adopt various reagents, amounts and conditions conventionally used in the art.
  • the solid-phase phosphoramidite synthesis may use the following conditions:
  • the deprotection conditions of the nucleoside monomer include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, the deprotection reagent can be selected One or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, and in some embodiments, dichloroacetic acid.
  • the molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support can be 2:1-100:1, and in some embodiments, 3:1-50:1 .
  • the coupling reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, and the molar ratio of the nucleic acid sequence linked to the solid support to the nucleoside monomer can be 1:1-1:50. In some embodiments, it is 1:5-1:15; the molar ratio of the nucleic acid sequence connected to the solid-phase carrier and the coupling reagent is 1:1-1:100, and in some embodiments, it is 1:50-1:80
  • the reaction time can be 200-3000 seconds, for example, 500-1500 seconds.
  • the coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole, for example 5-ethylthio- 1H-tetrazolium.
  • the capping reaction conditions include a temperature of 0-50°C, 15-35°C in some embodiments, a reaction time of 5-500 seconds, and 10-100 seconds in some embodiments, and the selection of capping reagents is the same as described above.
  • the molar ratio of the total amount of capping reagent to the nucleic acid sequence linked to the solid phase carrier is 1:100-100:1, and in some embodiments, 1:10-10:1.
  • the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole
  • the molar ratio of acetic anhydride, N-methylimidazole and the nucleic acid sequence linked to the solid-phase carrier can be 1:1:10-10: 10:1, in some embodiments 1:1:2-2:2:1.
  • the oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, and in some embodiments 5-50 seconds, the oxidizing reagent is iodine in some embodiments. (In some embodiments, it is provided in the form of iodine water).
  • the molar ratio of the oxidizing reagent to the nucleic acid sequence linked to the solid support in the coupling step may be 1:1-100:1, and in some embodiments, 5:1-50:1.
  • the oxidation step is replaced by sulfurization.
  • the vulcanization reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, and in some embodiments 100-1000 seconds, the vulcanization reagent is hydrogenated in some embodiments. Xanthan.
  • the molar ratio of the vulcanizing reagent to the nucleic acid sequence linked to the solid support in the coupling step is 10:1 to 1000:1, and in some embodiments, 10:1 to 500:1.
  • the method also includes separating the sense strand and antisense strand of the siRNA.
  • the separation method is well known to those skilled in the art, and generally includes cutting the synthesized nucleotide sequence from the solid support, removing the protective group on the base and phosphate, purification and desalting.
  • Cleaving the synthesized nucleotide sequence from the solid-phase carrier and removing the protective groups on the base and phosphate can be carried out in accordance with the conventional cleavage and deprotection methods in siRNA synthesis.
  • the obtained nucleotide sequence to which the solid-phase carrier is attached is contacted with concentrated ammonia water.
  • the concentrated ammonia water may be 25-30% by weight of ammonia water, and the amount of concentrated ammonia water may be 0.2ml/ ⁇ mol-0.8ml/ ⁇ mol compared with the target siRNA sequence.
  • the method further includes contacting the nucleotide sequence removed from the solid phase carrier with triethylamine trihydrofluoride to remove the 2'-TBDMS protection.
  • the corresponding nucleotide in the obtained target siRNA sequence has a free 2'-hydroxyl group.
  • the amount of pure triethylamine trihydrofluoride can be 0.4ml/ ⁇ mol-1.0ml/ ⁇ mol. In this way, siRNA that can be used in the transfection complex of the present disclosure can be obtained.
  • a preparative ion chromatography purification column can be used to complete the purification of nucleic acid through gradient elution of NaBr or NaCl; after the product is collected and combined, a reversed-phase chromatography purification column can be used for desalination.
  • the non-bridging oxygen atom or sulfur atom in the phosphodiester bond between the nucleotides or the phosphorothioate bond is basically combined with sodium ions, and the obtained siRNA basically exists in the form of sodium salt.
  • a well-known ion exchange method can be used to replace the sodium ions with hydrogen ions and/or other cations (for example, other alkali metal ions or ammonium ions) to obtain other forms of siRNA.
  • nucleic acid purity can be detected by ion exchange chromatography, and molecular weight can be determined by liquid mass spectrometry (LC-MS).
  • LC-MS liquid mass spectrometry
  • the annealing method is also well known to those skilled in the art.
  • the synthesized sense strand (S chain) and antisense strand (AS chain) can be simply mixed in an equimolar ratio, heated to 70-95°C in water for injection, and then cooled at room temperature to form a double bond through hydrogen bonding. Chain structure.
  • siRNA that can be used in the transfection complex of the present disclosure can be obtained.
  • the synthesized siRNA can be characterized by, for example, molecular weight detection by using, for example, liquid chromatography-mass spectrometry, and the sequence of the synthesized siRNA is determined to be the sequence of the desired siRNA. , For example, is one of the sequences listed in Table 1.
  • the content of the bioactive agent may vary within a relatively large range, as long as there is no harm to the subject.
  • the weight ratio of bioactive molecules to total lipids in the transfection complexes of the present disclosure is in the range of about 1:1-1:200, wherein the total lipids are the key lipids. (Ie, the amine-containing transfection reagent of the present disclosure), the sum of auxiliary lipids and pegylated lipids.
  • the weight ratio is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:1 to about 1:20, from about 1:2 to About 1:18, from about 1:3 to about 1:17, from about 1:4 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or from In the range of about 1:6 to about 1:10, for example, the weight ratio of the bioactive agent to the total lipid is about 1:3, 1:4, 1:5, 1:6, 1:7, 1: 8, 1:9, or 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.
  • the transfection complex particles formed by the siRNA and the above-mentioned amine-containing transfection reagent have an average diameter (ie, an apparent average particle size) of about 30 nm to about 200 nm, usually About 40nm to about 135nm, in some embodiments, the average diameter of the transfection complex particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, for example, the transfection complex particles The average diameter of the dye composite particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.
  • the transfection complex particles of the present disclosure have uniform dispersibility (PDI). In some embodiments, the transfection complex particles of the present disclosure have a dispersibility of 0.03-0.5. In some embodiments, the transfection complex particles of the present disclosure have a dispersibility of 0.05-0.35. In some embodiments, the transfection complex particles of the present disclosure have a dispersibility of 0.07-0.166.
  • PDI uniform dispersibility
  • the transfection complex particles of the present disclosure have a dispersibility of 0.03-0.5. In some embodiments, the transfection complex particles of the present disclosure have a dispersibility of 0.05-0.35. In some embodiments, the transfection complex particles of the present disclosure have a dispersibility of 0.07-0.166.
  • the transfection complex of the present disclosure has an encapsulation efficiency of 70.3% to 99.9%. In some embodiments, the transfection complex of the present disclosure has an encapsulation efficiency of 75.0%-99.0%. In some embodiments, the transfection complex of the present disclosure has an encapsulation efficiency of 79.3%-98.15%.
  • each component of the transfection complex of the present disclosure may exist independently when sold, and may exist in the form of a liquid formulation when used.
  • the preparation method of the transfection complex provided in the present disclosure includes providing a solution containing a key lipid, and incubating the solution containing the key lipid, the key lipid being the amine-containing transfection reagent provided in the present disclosure.
  • the key lipid-containing solution also contains auxiliary lipids and pegylated lipids.
  • auxiliary lipids and pegylated lipids are as described above.
  • the method further includes concentrating or diluting the liposome formulation after the incubation. In some embodiments, the method further includes removing impurities and/or sterilizing.
  • the method further includes mixing the key lipid-containing solution with the bioactive agent-containing solution before the incubation.
  • the type and content of the bioactive agent are as described above.
  • the solvents and their amounts in the solution containing the key lipids and the solution containing the biologically active agent are all conventional choices.
  • the incubation conditions can be conventional conditions.
  • Concentration or dilution can be performed before, after, or at the same time as removing impurities.
  • the method of removing impurities and the method of sterilization can adopt conventional methods.
  • the method of removing impurities can use a tangential flow system, a hollow fiber column, ultrafiltration under 100K Da conditions, and the ultrafiltration exchange solution is phosphate buffered saline (PBS) with pH 7.4. It can be filtered and sterilized on a 0.22 ⁇ m filter.
  • PBS phosphate buffered saline
  • the transfection complex provided in the present disclosure can be prepared according to the following method:
  • the amine-containing transfection reagent (key lipid), auxiliary lipid, and pegylated lipid provided in the present disclosure are mixed in alcohol according to the above-mentioned molar ratio to obtain a solution containing the key lipid; the amount of alcohol is such that it contains The total mass concentration of the key lipid solution is 2-25 mg/mL, for example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid around room temperature. In some embodiments, the alcohol is selected from ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 200, and polyethylene glycol.
  • One or more of alcohol 300 and polyethylene glycol 400, for example, may be ethanol.
  • the concentration of the buffer salt solution is 0.05-0.5M, such as 0.1-0.2M, adjust the pH of the buffer salt solution to 4.0-5.5, such as 5.0-5.2, and the amount of the buffer salt solution is such that the concentration of siRNA does not exceed 0.6 mg /mL, for example, 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, it may be sodium acetate and/or potassium acetate.
  • the solution containing the key lipids and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation.
  • the volume ratio of the solution containing the key lipid to the siRNA aqueous solution is 1:(2-5), for example, it may be 1:4.
  • the liposome preparation after the incubation is concentrated or diluted to remove impurities and sterilize to obtain the transfection complex provided by the present disclosure.
  • the physical and chemical parameters of the transfection complex are pH 6.5-8, and the encapsulation efficiency is not low At 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be pH 7.2-7.6, and the encapsulation rate is not less than 90%.
  • the average particle size (average particle diameter) is 60-100nm, the polydispersity index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • transfection complex of the present disclosure in the preparation of a medicament for the treatment and/or prevention of pathological conditions or diseases caused by the expression of specific genes in cells.
  • the present disclosure provides a method for treating and/or preventing a pathological condition or disease caused by the expression of a specific gene in a cell.
  • the method includes transferring an effective amount of the bioactive agent of the present disclosure.
  • the dye complex is given to subjects in need.
  • the mechanism of RNA interference can be used to treat and/or prevent the expression of specific genes in cells.
  • the pathological condition or purpose of the disease. Therefore, the transfection complex of the present disclosure can be used to treat and/or prevent pathological conditions or diseases caused by the expression of a specific gene in a cell, or be used in preparation for the treatment and/or prevention of a disease caused by the expression of a specific gene in a cell. Drugs that cause pathological conditions or diseases.
  • the specific gene may be an endogenous gene expressed in the cell, or a pathogen gene that reproduces in the cell.
  • the specific gene is selected from ApoB, ApoC3, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, HCV genes.
  • the biologically active agent is selected from the biologically active agent capable of specifically binding to the mRNA corresponding to the above-mentioned gene, such as siRNA targeting the mRNA corresponding to the above-mentioned gene.
  • the specific gene is selected from hepatitis B virus gene, angiopoietin-like protein 3 gene, or apolipoprotein C3 gene.
  • the disease is selected from chronic liver disease, hepatitis, liver fibrotic disease, liver proliferative disease and dyslipidemia.
  • the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.
  • the transfection complexes provided in the present disclosure can also be used to treat other diseases, including diseases characterized by unwanted cell proliferation, blood diseases, metabolic diseases, and diseases characterized by inflammation.
  • the proliferative disease can be a benign or malignant disease, such as cancer or cell tumor.
  • Hematological or inflammatory diseases may be diseases involving coagulation factors, complement-mediated inflammation or fibrosis.
  • Metabolic diseases include dyslipidemia and irregularities in glucose regulation.
  • the disease is treated by administering one or more oligonucleotides that have a high degree of homology with the gene sequence of the participating cells.
  • administration/administration refers to a method or approach that allows the transfection complex of the present disclosure to be at least partially positioned at a desired site to produce a desired effect. Placed into the subject.
  • routes of administration suitable for the method of the present disclosure include local administration and systemic administration. In general, local administration results in delivery of more transfection complexes to a specific site than the subject's systemic circulation; while systemic administration results in delivery of the transfection complexes of the present disclosure to the subject's systemic circulation.
  • the subject can be administered to the subject by any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, and transdermal administration. Medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including oral administration and sublingual administration).
  • the frequency of administration can be once or more daily, weekly, every two weeks, every three weeks, every month, or every year.
  • the dosage of the transfection complex described in the present disclosure may be a conventional dosage in the art, and the dosage may be determined according to various parameters, especially the age, weight, and sex of the subject. May be in cell cultures or experimental animals Determination Toxicity and therapeutic efficacy by standard pharmaceutical procedures, e.g. assays and ED 50 dose (the amount of the reaction finger to cause 50% of the maximum intensity of the reaction of the LD 50 (so that dose lethal to 50% of the population died), In the qualitative response, it refers to the dose that can cause a positive response in 50% of the subjects).
  • the range of human doses can be derived based on data obtained from cell culture analysis and animal studies.
  • the siRNA dosage may be 0.001-50 mg/kg body weight, in some embodiments 0.01-10 mg/kg body weight, in some embodiments 0.05-5 mg/kg body weight, in some embodiments 0.1-3 mg /kg body weight.
  • the present disclosure provides a method for inhibiting the expression of a specific gene in a cell, the method comprising contacting an effective amount of the transfection complex of the present disclosure with the cell, and introducing the transfection complex of the present disclosure into the cell.
  • the cell promotes the biologically active agent in the transfection complex to enter the cell to inhibit the expression of a specific gene.
  • the cell is a hepatocyte.
  • the liver cells can be selected from SMMC-7721, HepG2, Huh7 liver cancer cell lines or isolated primary liver cells.
  • the cell is an isolated liver primary cell.
  • the amount of the bioactive agent, especially the functional oligonucleotide, in the provided transfection complex can be easily determined by those skilled in the art according to the desired effect .
  • the functional oligonucleotide is siRNA
  • the amount of siRNA in the provided transfection complex containing siRNA is generally such an amount that the amount of the siRNA is sufficient to reduce the expression of the target gene , And result in an extracellular concentration of 1 pM to 1 ⁇ M, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to about 5 nM at the surface of the target cell.
  • the amount required to achieve this local concentration will vary with various factors including, for example, the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, and the delivery route (local or systemic) .
  • the concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.
  • the transfection complexes provided in the present disclosure have unexpectedly low toxicity and higher or at least comparable activity in vivo.
  • the siRNA-containing transfection complex provided by the present disclosure shows an excellent target gene suppression effect.
  • the transfection complex provided by the present disclosure containing siRNA directed against ApoB gene expression exhibits excellent characteristics of inhibiting ApoB gene expression: while having low off-target effects, the transfection complex can be at 1 mg/kg It inhibited 93.89%-94.30% of ApoB gene expression in BALB/c mouse liver at the same dosage.
  • the transfection complex containing siRNA provided in the present disclosure can also effectively reduce the blood lipid level in BALB/c mice, and at a dose of 1 mg/kg, it can approach the situation of transfection with the transfection complex of the prior art.
  • the inhibition level of serum CHO and TG can even reach a higher level of inhibition.
  • the siRNA-containing transfection complex provided by the present disclosure also exhibits unexpectedly low acute liver toxicity, especially when compared with the prior art transfection complex, it is observed in general anatomy The obtained organ lesions occur at higher concentrations, and the maximum tolerated dose (MTD) is increased to at least 1.4 times, or even 2.9 times, of the transfection complex of the prior art.
  • the experimental animals given the transfection complex of the prior art have shown obvious toxic pathological phenomena, such as liver discoloration and ascites, the general anatomy of the experimental animals given the same dose of the transfection complex of the present disclosure unexpectedly did not show Obviously abnormal. Therefore, the amine-containing transfection reagents and transfection complexes of the present disclosure show excellent potential in the in vivo transfection and delivery of biologically active agents, especially functional oligonucleotides.
  • the present disclosure provides a kit comprising an effective amount of the transfection complex of the present disclosure.
  • kits described in the present disclosure can provide transfection complexes in one container.
  • the kits described in the present disclosure may include a container that provides pharmaceutically acceptable excipients.
  • the kit may also contain other ingredients, such as stabilizers or preservatives.
  • the kit described in the present disclosure may contain at least one other therapeutic agent in a container other than the container in which the bioactive agent described in the present disclosure is provided.
  • the kit may provide each or more components of the transfection complex in different containers.
  • the kit may include instructions for mixing each or more components of the transfection complex to obtain the transfection complex of the present disclosure.
  • the transfection complex or its components can be provided in any form, such as a liquid form, a dried form or a lyophilized form.
  • the transfection complex or its components are substantially pure and/or sterile.
  • sterile water can be provided in the kit of the present disclosure.
  • CD1 mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.;
  • BALB/c mice 6-8 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • step (1-1) To 11g N5_1 (21.4mmol, 1.0eq) obtained in step (1-1) was added 12.2g N-(2,3-epoxypropyl)phthalamide (60.0mmol, 2.8eq), 4.1g two Isopropylethylamine (DIEA, 32.1mmol, 1.5eq) and 150ml DMF. React at 120°C for 6 hours and continue to react overnight. The reaction solution was cooled to room temperature and poured into 1 L of water. The aqueous layer was discarded, 50 ml of water and 30 ml of dichloromethane (DCM) were added to the residue, the organic layer was separated, and the aqueous layer was extracted once with 20 ml of DCM.
  • DCM dichloromethane
  • amine-containing Transfection reagent 1 (280mg, purity 92.0%), amine-containing transfection reagent 2 (300mg, purity 91.7%), and amine-containing transfection reagent 3 (110mg, purity 85.6%), of which the purity was detected by HPLC and by mass spectrometry Molecular weight, confirm that amine-containing transfection reagent 1, amine-containing transfection reagent 2, and amine-containing transfection reagent 3 are amine-containing transfection reagents having the structure represented by formula (101), (102), or (103), respectively.
  • SiRNA1 and siRNA2 as shown in Table 2-1 were synthesized respectively.
  • siRNA1 and siRNA2 are completely the same.
  • the specific synthesis method is as follows:
  • Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, and oxidation.
  • the synthesis conditions are given as follows:
  • the nucleoside monomer is provided in an acetonitrile solution with a concentration of 0.1M.
  • the conditions of the deprotection reaction in each step are the same, that is, the temperature is 25°C, the reaction time is 70 seconds, and the deprotection reagent is dichloroacetic acid in dichloromethane (3% v/v), the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.
  • each step of the coupling reaction is the same, including the temperature of 25°C, the molar ratio of the nucleic acid sequence connected to the solid-phase carrier to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid-phase carrier and the coupling reagent The ratio is 1:65, the reaction time is 600 seconds, and the coupling reagent is a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazolium.
  • the capping conditions for each step are the same, including a temperature of 25°C and a reaction time of 15 seconds.
  • the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, where Cap1 and Cap2 are capping reagent solutions, and Cap1 is a 20% (v/v) N-methylimidazole pyridine/acetonitrile mixed solution, pyridine
  • the volume ratio to acetonitrile is 3:5;
  • Cap2 is 20% (v/v) acetonitrile solution of acetic anhydride;
  • the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid support is acetic anhydride: N-methylimidazole: solid
  • the nucleic acid sequence linked on the phase carrier 1:1:1.
  • the oxidation reaction conditions for each step are the same, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidizing reagent of 0.05M iodine water.
  • the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling step is 30:1.
  • the cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected with the carrier is added to ammonia water with a concentration of 25% by weight, the amount of ammonia water is 0.5ml/ ⁇ mol, the reaction is carried out at 55°C for 16h, the liquid is removed, and it is concentrated to dryness in vacuo. After ammonia treatment, relative to the amount of single-stranded nucleic acid, dissolve the product with 0.4ml/ ⁇ mol N-methylpyrrolidone, and then add 0.3ml/ ⁇ mol triethylamine and 0.6ml/ ⁇ mol triethylamine trihydrofluoride to remove In addition to the 2'-TBDMS protection on ribose.
  • eluent A 20mM sodium phosphate (pH 8.1)
  • eluent B 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1)
  • elution gradient: eluent A: eluent B 100:0-50:50 gradient elution.
  • the product eluates are collected and combined, and desalted using a reversed-phase chromatography purification column.
  • the specific conditions include using a Sephadex column for desalting, the filler is Sephadex G25, and elution with deionized water.
  • Detection use ion exchange chromatography (IEX-HPLC) for detection; use liquid mass spectrometry (LC-MS) to analyze molecular weight.
  • IEX-HPLC ion exchange chromatography
  • LC-MS liquid mass spectrometry
  • the siRNA sense strand SS is obtained in this step.
  • Detection Purity is detected by ion exchange chromatography (IEX-HPLC); molecular weight is analyzed by liquid-mass spectrometry (LC-MS).
  • the SS chain and AS chain were separately dissolved in water for injection to obtain a 40mg/ml solution. They were mixed in an equimolar ratio, heated at 50°C for 15 minutes, and cooled at room temperature to obtain siRNA1 and siRNA2.
  • LC-MS Liquid Chromatography-Mass Spectrometry
  • amine-containing transfection reagent solution Preparation of amine-containing transfection reagent solution.
  • the amine-containing transfection reagent 1-3 and the comparative amine-containing transfection reagent LC8, cholesterol, and PEGylated lipid (1,2-two) obtained above were prepared separately. Palmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000(DPPE-PEG)) was mixed with ethanol at a molar ratio of 52:20:28 to obtain a mixture containing
  • the concentration of the four amine-containing transfection reagent ethanol solutions of Amine Transfection Reagents 1-3 and LC8, and the four amine-containing transfection reagent ethanol solutions are all 6.50 ⁇ M.
  • the comparative amine-containing transfection reagent LC8 is a cationic lipid 87 prepared in accordance with Example 12 of Chinese Patent CN 103380113B, and the amine-containing transfection reagent has a structure as shown in formula (901):
  • siRNA1 Dissolve siRNA1 in a 200mM sodium acetate (pH 5.2) solution to make the concentration of siRNA1 0.4mg/ml to obtain an aqueous solution of siRNA1 sodium acetate.
  • amine-containing transfection reagent solution Prepare the amine-containing transfection reagent solution according to the same method as in 2) A), except that the amine-containing transfection reagents used are the amine-containing transfection reagent 1-2 and the comparative amine-containing transfection reagent LC8, respectively.
  • siRNA2 is used instead of siRNA1 to obtain the siRNA2 sodium acetate aqueous solution.
  • the transfection complex was prepared according to the same method as in 2) C), except that the three amine-containing transfection reagent ethanol solutions containing amine-containing transfection reagent 1-2 and LC8 were used instead of the four kinds of amine-containing transfection reagent ethanol solutions obtained above.
  • the encapsulation efficiency was detected by the RiboGreen method, and the reagents used (Quant-iT TM RNA Reagent and Kit) were purchased from Thermo Fisher (Invitrogen) Company, with the item number R11490. Detect the fluorescence intensity of siRNA in the sample according to the operating steps of the instructions, and then calculate the encapsulation efficiency according to the method described in the literature (J. Heyes et.al, Journal of Controlled Release, 107(2005):276-287):
  • Encapsulation rate [(fluorescence intensity of Triton treatment group-fluorescence intensity of no Triton treatment group)/fluorescence intensity of Triton treatment group] ⁇ 100%
  • the light scattering method (ZetaPALS, Brookhaven Instruments) was used to detect the apparent average particle size and dispersion according to the instrument manual, and the siRNA concentration was measured by NANO DROP 2000 (Thermo).
  • mice Female BALB/c mice aged 6-8 weeks and weighing 18-25g were randomly divided into 13 groups, which were recorded as negative control group (1 group, 1 ⁇ PBS solution) and positive control group (3 groups, respectively) Different doses of contrast transfection complex 1) and the experimental group (3 ⁇ 3 groups, respectively given different doses of transfection complex 1, transfection complex 2 or transfection complex 3), including 6 negative control groups There are 5 mice in each group of mice, positive control group and experimental group.
  • the mode of administration is a single tail vein administration, the dosage (calculated as siRNA) is 1mg/kg, 0.5mg/kg, 0.1mg/kg, and the administration volume is 10mL/kg. Blood was collected from the orbit 24 hours after the administration, and then the animals were sacrificed, the animals were grossly dissected and the organs were observed, the liver tissues were collected, and stored separately with RNA later (Sigma Aldrich) for testing;
  • RNAVzol for each of the aforementioned positive control group and experimental group, add 1ml/g RNAVzol to the liver tissue to be tested, add steel balls and homogenize in a Tissuelyset II type automatic tissue homogenizer for 1 minute; Add 0.2ml of chloroform to the sample, shake for 15 seconds, and let stand for 3 minutes; centrifuge at 12000 rpm for 15 minutes at 4°C to collect the supernatant; add 0.5 mL of isopropanol to the supernatant, mix well, and let stand at room temperature 10 minutes; centrifuge at 12000rpm for 10 minutes at 4°C, discard the supernatant; add 1mL 75% alcohol to wash the pellet, centrifuge at 12000rpm for 5 minutes at 4°C, discard the supernatant; dry, add 150 ⁇ L DEPC water Dissolve. Obtain the total RNA extracted from mouse liver tissue.
  • NANO DROP 2000 (Thermo) was used to measure the concentration of the extracted total RNA, and 1% agarose gel electrophoresis (Agarose, OXOID Lot: 1315449) was performed to detect the quality of the extracted total RNA.
  • RNA electrophoresis conditions 1% gel, 0.8 ⁇ g sample, Marker: 1Kb DNA ladder (TRANS, Lot#I11112, Code: #BM201), 90V, 20 minutes.
  • the product was subjected to 1% agarose gel electrophoresis (Agarose, OXOID Lot: 1315449) to detect the amplification quality. Electrophoresis conditions: 1% gel, 6 ⁇ L loading, Marker: 100Kb DNA ladder (TRANS, Lot#K21022, Code:#BM301), 120V, 25 minutes.
  • ApoB mRNA expression level (ApoB mRNA expression level in the test group/GAPDH mRNA expression level in the test group)/(ApoB mRNA expression level in the control group/GAPDH mRNA expression level in the control group) ⁇ 100%
  • Inhibition rate [1-(ApoB mRNA expression in the test group/GAPDH mRNA expression in the test group)/(ApoB mRNA expression in the control group/GAPDH mRNA expression in the control group)] ⁇ 100%, where the inhibition rate refers to the inhibition rate of the transfection complex on ApoB mRNA expression
  • Fig. 1 and Fig. 2 The inhibitory rates of the transfection complex prepared with different doses of the compound of the present disclosure and the comparative transfection complex on the expression level of ApoB mRNA in liver tissues in BALB/c mice are shown in Fig. 1 and Fig. 2.
  • transfection complex 2 in mice, the ApoB mRNA inhibitory activity of transfection complexes 1 and 2 is close to or even better than that of comparative transfection complex 1 at the same dose.
  • transfection complex 2 showed an unexpectedly higher ApoB mRNA inhibition rate compared with comparative transfection complex 1.
  • the mRNA inhibition rate of Transfection Complex 2ApoB is 126.3% of that of Comparative Transfection Complex 1.
  • Example 4 Acute toxicity test of transfection complex 4-5 and comparative transfection complex 2 in CD1 mice
  • This example is used to detect the toxicity of the transfection complex 4-5 and the comparative transfection complex 2 obtained in Example 2 in CD1 mice.
  • CD1 mice aged 6-8 weeks and weighing 18-25g were randomly divided into groups, and they were marked as positive control group (comparative transfection complex 2) and experimental group (transfection complex 4 and transfection complex 5). There are 6 mice in each group (3 male and female, labeled M and F respectively). According to the dose values listed in Table 4, each group of mice was given contrast transfection complex 2, transfection complex 4 or transfection complex 5, respectively.
  • M and F respectively represent the sex of the mouse, and the numbers before M and F represent the number of deaths.
  • “1F” means that one female mouse died
  • “3M, 3F” "Means that all three male mice and three female mice died.
  • the number group with a slash “/” indicates the number of mice that survived and local discoloration of the liver
  • the number after the slash "/” indicates the number of surviving mice in this group of tested mice
  • the number before the slash "/” indicates the number of mice with local liver discoloration among the surviving mice in this group.
  • the preceding text indicates the degree of liver discoloration. For example, "local liver discoloration 2/6" indicates survival to the last Of the 6 mice, 2 developed local discoloration of the liver.
  • MTD maximum tolerated dose
  • Comparative transfection complex 2 9.5-10.8mg/kg
  • the transfection complex of the present disclosure shows lower acute toxicity at the same dose, and in terms of the maximum tolerated dose, the transfection complex of the present disclosure unexpectedly shows significantly more
  • the high tolerance value, especially the transfection complex 5 showed a maximum tolerated dose value of about 1.4-2.9 times compared with the comparison transfection complex 2.
  • the comparative transfection complex has shown a high proportion of hepatotoxicity, while at the same dose, the experimental animals given the transfection complex of the present disclosure have liver toxicity.
  • the necropsy showed unexpectedly lower toxicity, especially the experimental animals given the same dose range of transfection complex 5 did not show any obvious abnormalities in the liver necropsy.
  • the amine-containing transfection reagent of the present disclosure unexpectedly exhibits significantly lower acute toxicity when the delivery efficiency is close to or even better than that of the prior art compounds.

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Abstract

本公开涉及一种含胺转染试剂和其制备方法以及包含该含胺转染试剂的转染复合物。该含胺转染试剂为式(I)所示化合物或其药学上可接受的盐，含有该含胺转染试剂的转染复合物具有较佳的生物活性和出人意料的低毒性。

Description

一种含胺转染试剂和制备方法及转染复合物 技术领域
本公开涉及生物医药领域，特别涉及一种可用于递送生物活性剂的含胺转染试剂、该含胺转染试剂的制备方法及包含该含胺转染试剂的转染复合物。
背景技术
脂质体(liposome)是一种含有脂质的双层结构的人工膜，一般包含磷脂、胆固醇等成分，在溶液中形成纳米脂质颗粒。脂质体可以用于包裹和递送药物，将药物递送入细胞内部。脂质体通常可以用于递送小分子药物，目前已经有多个脂质体小分子药物上市。此外，脂质体也可以用于包裹核酸，将核酸转染到细胞内，或递送到靶组织并进入细胞。本领域中通常使用含胺脂质化合物(即转染试剂)递送核酸分子，例如，中国专利CN 103380113B中描述了多种含胺脂质化合物。这些脂质化合物中的胺类基团与DNA或RNA具有强的静电结合作用，因而可用于核酸分子的递送。然而，这种含胺脂质化合物有时会带来一定的毒性。因此如何在提高脂质体递送核酸药物活性的同时降低毒性，仍是本领域需要解决的问题。
发明内容
本公开提供了一种含胺转染试剂，该转染试剂以及包含该转染试剂的转染复合物具有较佳的生物活性和较低毒性，适合用于生物活性剂，特别是核酸药物的体内递送。
在一些方面，本公开提供了一种含胺转染试剂，该含胺转染试剂为式(I)所示的化合物或其药学上可接受的盐：
Figure PCTCN2019125165-appb-000001
其中，
Y 1选自C2-C10的亚烷基或具有取代基的C2-C10亚烷基；
每个Y 2相同或不同，独立地选自C2-C6的亚烷基或具有取代基的C2-C6亚烷基；
每个R 1相同或不同，独立地选自H或式(I-i)所示的基团，每个R 2独立地为式(I-ii)所示的基团，
Figure PCTCN2019125165-appb-000002
其中，每个R a和每个R b各自独立地选自C6-C20直链烷基，每个R c各自独立地选自非胺类亲水性基团中的一种，
Figure PCTCN2019125165-appb-000003
表示基团共价连接的位点。
在一些方面，本公开还提供了一种含胺转染试剂的制备方法，该含胺转染试剂为式(I)所示化合物或其药学上可接受的盐，该方法包括：在有机溶剂中，在偶联试剂存在下和偶联反应条件下，以及在足以生成式(I)所示化合物的条件下，将式(301)所示的化合物与式(701)所示的化合物接触，分离出式(I)所示化合物；
Figure PCTCN2019125165-appb-000004
式(301)和式(701)中，基团Y 1、Y 2、R a、R 2的定义与取值范围如前所述，Z 701为离去基团。
在一些方面，本公开还提供一种转染复合物，该转染复合物含有关键脂质，所述关键脂质为本公开提供的上述含胺转染试剂。
在一些方面，本公开还提供本公开的转染复合物在制备用于治疗和/或预防细胞中特定基因的表达而引起的病理状况或疾病的药物中的用途。
在一些方面，本公开还提供一种治疗和/或预防由特定基因的表达而引起的病理状况或疾病的方法，所述方法包括向患有该病理状况或疾病的受试者给予本公开的转染复合物。
在一些方面，本公开还提供一种抑制细胞中特定基因表达的方法，该方法包括将本公开的转染复合物与所述细胞进行接触。
在一些方面，本公开还提供一种试剂盒，该试剂盒包含本公开提供的转染复合物。
附图说明
图1示出了实施例3所示的以本公开化合物制备的转染复合物和对比转染复合物在剂量为1mg/kg以及0.5mg/kg时，在BALB/c小鼠体内对肝组织中ApoB mRNA表达水平的抑制率。
图2示出了实施例3所示的以本公开化合物制备的转染复合物和对比转染复合物在剂量为0.1mg/kg时，在BALB/c小鼠体内对肝组织中ApoB mRNA表达水平的抑制率。
图3示出了实施例3所示的以本公开化合物制备的转染复合物和对比转染复合物在BALB/c小鼠体内对血清中总胆固醇(CHO)浓度的影响。
图4示出了实施例3所示的以本公开化合物制备的转染复合物和对比转染复合物在BALB/c小鼠体内对血清中甘油三酯(TG)浓度的影响。
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。本公开的特点和优点将通过以下具体实施方式得到更好的理解。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。
定义
如在说明书和所附的权利要求书中所使用的，除非有相反的特定说明，否则本公开的术语具有如下表示的意义：
如本公开所使用的，不介于两个字母之间或两个符号之间的短横(“-”)是用于指示取代基连接点的位置。例如：-C 1-C 10烷基-NH 2通过C 1-C 10烷基而连接。
如本公开所使用的，“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。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。
术语“卤素”是指氟、氯、溴或碘，优选氟或氯。
“受试者”一词，如本公开所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。在一些实施方式中，受试者指患有特定疾病的人类患者。
含胺转染试剂
在一方面，本公开提供了一种用于递送生物活性剂的含胺转染试剂，所述含胺转染试剂通过与作为药物活性成分的生物活性剂形成转染复合物(通常以脂质体的形式存在)而递送所述生物活性剂进入细胞。其中，“生物活性剂”包括但不限于能够通过本公开的转染复合物递送的、具有治疗和/或预防活性的药物分子、药物组合物、药物复合物、药物前体中的一种或多种，或者它们中任意一种或多种的药学上可接受的盐。在一些实施方式中，生物活性剂包括小分子药物、功能性寡核苷酸或药物蛋白、或者它们的药物前体或药学上可接受的盐，特别是本公开在以下描述的那些。
在一些实施方式中，本公开提供了一种含胺转染试剂，该含胺转染试剂为式(I)所示结构的化合物或其药学上可接受的盐：
Figure PCTCN2019125165-appb-000005
其中，
Y 1选自C2-C10亚烷基或具有取代基的C2-C10亚烷基；
每个Y 2相同或不同，独立地选自C2-C6的亚烷基或具有取代基的C2-C6亚烷基；
每个R 1相同或不同，独立地选自H或式(I-i)所示的基团，每个R 2独立地为式(I-ii)所示的基团，
Figure PCTCN2019125165-appb-000006
其中，每个R a和每个R b各自独立地选自C6-C20直链烷基，每个R c各自独立地选自非胺类亲水性基团中的一种，
Figure PCTCN2019125165-appb-000007
表示基团共价连接的位点。
在本公开的一些实施方式中，Y 1是C2-C10直链亚烷基。在一些实施方式中，Y 1是C3-C5直链亚烷基。在一些实施方式中，Y 1为亚丁基。术语“亚烷基”为由碳原子和氢原子组成的直链或支链的二价饱和烃基，位于两个其他化学基团之间并用于连接这两个其他化学基团。
在一些实施方式中，Y 2是具有一个羟基取代基的C2-C6亚烷基。在一些实施方式中，每个Y 2独立地是具有一个羟基取代基的C2-C3亚烷基。在一些实施方式中，每个Y 2均是2-羟基亚丙基。
根据本公开提供的含胺转染试剂，每个R 2独立地为(I-ii)所示的基团，其中每个R b各自独立地选自C6-C20直链烷基，每个R c各自独立地选自非胺类亲水性基团中的一种。出人意料地，R c选自非胺类亲水性基团，在一种实施方式选自羟基时，本公开提供的含胺转染试剂不仅能够保持或提高递送效率和所递送的生物活性剂的活性，而且其毒性得到大大降低。在一些实施方式中，每个R c各自独立地选自羟基、巯基、羧基、磷酸基团或聚乙二醇基团。在一些实施方式中，每个R c均为羟基。在一些实施方式中，每个R b独立地为C8-C16直链烷基。在一些实施方式中，所有R b均相同，例如每个R b均为十二烷基。
在一些实施方式中，每个R 1彼此独立地选自H或者由(I-i)所示的基团。其中，两个R 1可以均为H，均为由(I-i)所示的基团，或者一个R 1为H另一个R 1为(I-i)所示的基团。在一些实施方式中，两个R 1均为H，在一些方式中，一个R 1为H，另一个R 1为(I-i)所示的基团，配合本公开所述R c，本公开提供的含胺转染试剂表现出更好的效果。在一些实施方式中，每个R a独立地选自C6-C20直链烷基。在一些实施方式中，每个R a独立地选自C10-C18直链烷基。在一些实施方式中，所有R a均相同，例如每个R a均是十五烷基。
在一些实施方式中，本公开的含胺转染试剂选自式(101)-(103)所示的化合物及其药学上可接受的盐中的一种或多种。
Figure PCTCN2019125165-appb-000008
Figure PCTCN2019125165-appb-000009
其中，药学上可接受的盐可以选自本领域常用的药学上可接受的盐，只要是所述盐处于合理的医学评价范围内，适用于与人体和动物的组织和器官接触，而不表现出额外的毒性、刺激、过敏反应等，并且与合理的益处/风险比一致。药学上可接受的盐可以是与有机酸和/或无机酸形成的药学上可接受的酸加成盐，例如乙酸盐、谷氨酸盐、乳酸盐、己二酸盐、苯甲酸盐、苹果酸盐、柠檬酸盐、扁桃酸盐、琥珀酸盐、甲磺酸盐、盐酸盐、氢溴酸盐、硫酸盐和磷酸盐中的一种或多种。
含胺转染试剂的制备
可以采用任意合理的合成路线制备式(I)所示化合物。
例如，在一些实施方式中，式(I)所示化合物的制备至少包括在有机溶剂中，在有机碱存在下和偶联反应条件下，以及在足以生成式(I)所示化合物的条件下，将式(301)所示的化合物与式(701)所示的化合物接触，分离出式(I)所示化合物；
Figure PCTCN2019125165-appb-000010
Figure PCTCN2019125165-appb-000011
式(301)和式(701)中，基团Y 1、Y 2、R a、R 2的定义与取值范围如前所述，Z 701为离去基团。
所述有机溶剂可以是卤代烷类、醚类、腈类和酰胺类溶剂中的一种或多种。在一些实施方式中，所述有机溶剂为二甲基甲酰胺(DMF)。相对于式(301)所示化合物，所述有机溶剂的用量可以是4-30L/mol，例如为6-20L/mol。所述有机碱可以是三级胺类有机碱，例如三乙胺或N,N-二异丙基乙胺(DIPEA)。所述有机碱与式(301)所示化合物的摩尔比为1:1-20:1，在一些实施方式中为3:1-10:1。式(701)所示化合物中，离去基团Z 701可以是本领域常用的离去基团，例如卤素、-OCOR、-OTs、-ONO 2或-OH。在一些实施方式中，离去基团Z 701是卤素。在一些实施方式中，离去基团Z 701是溴(Br)。式(701)所示化合物可由本领域技术人员容易地合成获得，或者可商购获得，例如，当离去基团Z 701是溴时，可由可商购获得的4-溴巴豆酸甲酯与不同碳链长度的脂肪醇出发，经本领域公知的酯交换方法容易地制备获得各种式(701)所示化合物。在本公开的实施方式中，依据所期望的产物式(I)所示化合物的结构，式(701)所示化合物与式(301)所示化合物的摩尔比可以是2:1-10:1，例如为2:1-6:1。所述偶联反应条件是指足以使离去基团Z 701离去并且使式(701)所示化合物与式(301)所示化合物发生反应形成共价连接的条件，而足以生成式(I)所示化合物的条件是指上述偶联反应充分地进行，其程度足以使得足够的式(701)所示化合物与式(301)所示化合物发生反应形成共价连接，从而生成式(I)所示化合物的反应。在一些实施方式中，反应可以在适当的温度如40-90℃下进行1-15小时。在一些实施方式中，所述反应在50℃下进行2h。在一些实施方式中，可通过薄层色谱法或HPLC对反应进程进行监测、通过特定指标例如反应混合物中的反应物/产物含量来确定反应终点。可使用任何合适的分离方法从反应混合物中分离式(I)所示化合物。所获得的反应产物可以是均符合式(I)所示结构、但R 1数量不同的化合物的混合物。因此，在一些实施方式中，可以在将该反应产物洗涤、干燥后，在柱色谱上，用洗脱剂梯度洗脱，收集具有单一化合物组成的洗脱液级分合并浓缩，分离出单一产物，其中可通过例如薄层色谱(TLC)、HPLC或LC-MS实时检测所收集的洗脱液是否具有单一组成，来确定所要合并的洗脱液级分。洗脱剂例如可以是二氯甲烷(DCM)和甲醇(MeOH)的混合溶液，二氯甲烷与甲醇的体积比为50:1-10:1。
在一些实施方式中，式(301)所示化合物的制备方法包括在有机溶剂中，在脱保护反应条件下，将式(302)所示化合物与脱保护试剂接触，分离出式(301)所示化合物。
Figure PCTCN2019125165-appb-000012
其中，R 2、Y 1、Y 2的定义和取值范围与前述相同，R 304是氨基保护基团。
所述有机溶剂可以是卤代烷类、醚类、醇类和酰胺类溶剂中的一种或多种；在一些实施方式中，所述有机溶剂为乙醇或甲醇。相对于式(302)所示化合物，所述有机溶剂的用量可以是4-30L/mol，例如为10-25L/mol。所述氨基保护基团R 304可以是本领域技术人员已知的各种氨基保护基团，例如叔丁氧羰基(BOC)、苄氧基(CBz)、苄基(Bn)、三甲基硅基、三氟乙酰基(CF 3CO)或乙酰基。在一些实施方式中，同一个氮原子上的两个R 304基团共同形成单个二价保护基团，如邻苯二甲酰基。所述脱保护试剂及其用量可根据氨基保护基团R 304而确定。在一些实施方式中，氨基保护基团是邻苯二甲酰基，此时脱保护试剂可以是例如水合肼，脱保护试剂与式(302)所示化合物的摩尔比可以是4:1-20:1，如5:1-8:1。反应可以在适当的温度，如40-90℃下进行1-15小时。在一些实施方式中，所述反应在溶剂回流条件下进行2h。在一些实施方式中，可通过色谱法或色谱-质谱联用法对反应进程进行监测。可使用任何合适的分离方法从反应产物中分离式(301)所示化合物。在一些实施方式中，可以将反应产物洗涤、干燥后，使用例如柱色谱对反应产生的式(301)所示化合物进行分离。在一些实施方式中，可直接将反应产物不作进一步处理而直接用于后续反应。
本领域技术人员可通过任意合理的路线制备获得式(302)所示化合物。在一些实施方式中，式(302)所示化合物中，Y 2是2-羟基-C2-C6亚烷基，式(302)所示化合物的制备方法包括在有机溶剂中，有机碱存在下和加成开环反应条件下，将式(303)所示化合物与式(703)所示化合物接触，分离出式(302)所示化合物。
Figure PCTCN2019125165-appb-000013
其中，R 2、Y 1、R 304的定义和取值范围与前述相同，Y 703是共价键或者C1-C4亚烷基。
所述有机溶剂可以是卤代烷类、醚类、醇类和酰胺类溶剂中的一种或多种；在一些实施方式中，所述有机溶剂为DMF。相对于式(303)所示化合物，所述有机溶剂的用量可以是4-20L/mol，例如为7-15L/mol。通过开环加成，式(703)所示化合物中的环氧乙烷基团开环形成与氨基连接的2-羟基亚乙基，从而与Y 703基团共同组成式(302)所示化合物中的Y 2基团。在一些实施方式中，式(703)所示化合物与式(303)所示化合物的摩尔比为2:1-4:1，例如可以为2.6:1-3.2:1。在一些实施方式中，所述有机碱是三级胺，例如三乙胺或N,N,-二异丙基乙胺。所述有机碱与式(303)所示化合物的摩尔比为1:1-10:1，在一些实施方式中为1.2:1-5:1。反应可以在适当的温度、如90-150℃下进行5-15小时。在一些实施方式中，所述反应在110-130℃下进行6-12h。可使用任何合适的分离方法从反应产 物中分离式(302)所示化合物。在一些实施方式中，可以在洗涤、干燥后，使用例如柱色谱对反应产生的式(302)所示化合物进行分离。在一些实施方式中，可直接将反应产物不作进一步处理而直接用于后续反应。
本领域技术人员可通过任意合理的路线制备获得式(303)所示化合物。在一些实施方式中，每个R c均为羟基，式(303)所示化合物的制备方法包括在有机溶剂中，在加成开环反应条件下，将式(304)所示的二胺类化合物与式(704)所示的环氧乙烷衍生物接触，分离出式(303)所示化合物。
Figure PCTCN2019125165-appb-000014
其中，Y 1和R b的定义和取值范围与前述相同。
所述有机溶剂可以是卤代烷类、醚类、醇类和酰胺类溶剂中的一种或多种；在一些实施方式中，所述有机溶剂为乙醇。相对于式(304)所示化合物，所述有机溶剂的用量可以是3-20L/mol，例如为4-10L/mol。通过开环加成，式(704)所示化合物中的环氧乙烷基团开环形成与氨基连接的2-羟基亚乙基，从而与R b基团共同组成式(303)所示化合物中的R 2基团。因此，在一些实施方式中，式(704)所示化合物与式(304)所示化合物的摩尔比为2:1-4:1，例如可以为2.1:1-2.6:1。反应可以在适当的温度，如25-70℃下进行2-10小时。在一些实施方式中，所述反应在50℃下进行4-6h。可使用任何合适的分离方法从反应产物中分离式(303)所示化合物。在一些实施方式中，可以在过滤、洗涤、干燥后，获得式(303)所示化合物。在一些实施方式中，可直接将反应产物不作进一步处理而直接用于后续反应。
上述式(703)所示化合物、式(704)所示化合物、式(304)所示化合物均可由本领域技术人员根据现有公开文献容易地制备获得，或商购获得。例如，当Y 1为亚丁基时，式(304)所示化合物为容易商购获得的1,4-丁二胺；当Y 703是亚甲基、且同一个氮原子上的两个R 304基团共同形成邻苯二甲酰基时，式(703)所示化合物是可商购获得的N-(2,3-环氧丙基)邻苯二甲酰胺；当R b为十二烷基时，式(704)所示化合物是容易商购获得的1,2-环氧十四烷。除非另有说明，本公开使用的其它原料也均是市售原料或根据现有公开容易制备获得的原料。
转染复合物
在另一方面，本公开提供了一种转染复合物，所述转染复合物包含本公开的含胺转染试剂。
本公开使用的术语“转染复合物”通常表示这样的组合物，该组合物用于将生物活性剂 (诸如核酸、药物蛋白或小分子)递送给细胞或体内或体外组织。
考虑到销售状态，不含生物活性剂的转染复合物可以作为产品单独生产和销售，因此，本公开提供的转染复合物可以包含关键脂质，所述关键脂质是指本公开所述的含胺转染试剂。
在一些实施方式中，该转染复合物还包含辅助脂质和/或聚乙二醇化脂质。所述关键脂质、辅助脂质和聚乙二醇化脂质之间的摩尔比可以在较大范围内变动。在一些实施方式中，该摩尔比为(15-100):(0-85):(0-50)；在一些实施方式中，该摩尔比为(19.7-80):(19.7-80):(0.3-50)；可选地，该摩尔比可以为(50-70):(20-40):(3-30)。
本公开使用的术语“辅助脂质”通常表示，在本公开提供的含胺转染试剂，即关键脂质之外，适用于制备和形成转染复合物的其它辅助性的脂质。合适的辅助脂质可以是本领域常用的辅助脂质，例如所述辅助脂质选自，但不限于，胆固醇、胆固醇类似物、胆固醇衍生物、甾醇类(包括植物甾醇、动物甾醇和藿烷类)、或者已知会实现或促进外源性生物活性剂向细胞或组织内部的引入的中性或阳离子脂质中的任一种或多种。在某些实施方案中，可以在本公开所述的转染复合物的配制中使用超过一种辅助脂质。可用于制备本公开所提供的转染复合物的示例性的、但非限制性的中性或阳离子脂质可以是选自下述的一种或多种脂质：BMOP(N-(2-溴乙基)-N,N-二甲基-2,3-二(9-十八烯基氧基)-丙铵溴化物)、DDPES(二棕榈酰磷脂酰乙醇胺5-羧基精胺基酰胺)、DSPC、CTAB:DOPE(鲸蜡基三甲基溴化铵(CATB)和DOPE的制剂)、POPC(1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱)、DOPE(二油酰磷脂酰乙醇胺)、DMG、DMAP(4-二甲基氨基吡啶)、DMPE(二肉豆蔻酰磷脂酰乙醇胺)、DOMG、DMA、DOPC(二油酰磷脂酰胆碱)、DMPC(二肉豆蔻酰磷脂酰胆碱)、DPEPC(二棕榈酰乙基磷脂酰胆碱)、DODAC(二油酰基二甲基氯化铵)、DOSPER(1,3-二油酰基氧基-2-(6-羧基精胺基)-丙基酰胺)、DOTMA(N-[1-(2,3-二油烯基氧基)丙基]-n,n,n-三甲基氯化铵)、DDAB(双十二烷基甲基溴化铵)、DOTAP(N-[1-(2,3-二油酰基氧基)丙基]-N,N,N-三甲基-铵甲基硫酸盐)、DOTAP·Cl、DC-chol(3,β-N,(N',N'-二甲基氨基乙烷)-氨甲酰基]胆固醇)、DOSPA(2-(精胺甲酰氨基)乙基)-N,N-二甲基-铵三氟乙酸盐)、DC-6-14(O,O'-双十四碳酰基-N-(α三甲基铵基乙酰基)二乙醇胺氯化物)、DCPE(二己酰磷脂酰乙醇胺)、DLRIE(二月桂基氧基丙基-3-二甲基羟基乙基溴化铵)、DODAP(1,2-二油酰基-3-二甲铵-丙烷)、乙基-PC、DOSPA(2,3-二油酰基氧基-N-[2-(精胺甲酰氨基乙基]-N,N-二甲基-1-丙铵三氟乙酸盐)、DOGS(双十八烷基酰氨基甘氨酰羧基精胺)、DMRIE(N-[1-(2,3-二肉豆蔻基氧基)丙基]-N,N-二甲基-N-(2-羟乙基)溴化铵)、DOEPC(二油酰基乙基-磷酸胆碱)、DOHME(N-[1-(2,3-二油酰基氧基)丙基]-N-[1-(2-羟乙基)]-N,N-二甲基碘化铵)、GAP-DLRIE:DOPE(N-(3-氨基丙基)-N,N-二甲基-2,3-二(十二烷氧基)-1-丙铵溴化物/二油烯基磷脂酰乙醇胺)、DPPC(二棕榈酰磷脂酰胆碱)、DOPG(1,2-二油酰基-sn-甘油-3-[磷酸-rac-(3-赖氨酰基(1-甘油))·Cl)、N-月桂酰基肌氨酸、(R)-(+)-柠檬烯、卵磷脂(和它们的衍生物)；磷脂酰乙醇胺(和它们的衍生物)；磷脂酰乙醇胺类、二油酰磷脂酰乙醇胺)、DPhPE(二植烷酰磷脂酰乙醇胺)、DPPE(二棕榈酰磷脂酰乙醇胺)、二棕榈酰磷脂酰乙醇胺、O-Chol(3β[1-鸟氨酰胺氨甲酰基]胆固醇)、POPE(棕榈酰油酰磷脂酰乙醇胺)和二硬脂酰磷脂酰乙醇胺；磷脂酰胆碱；磷脂酰胆碱类、DPPC(二棕榈酰磷脂酰胆碱)、POPC(棕榈酰油酰磷脂酰胆碱)和二硬脂酰磷脂酰胆碱；磷脂酰甘油；基于哌嗪的阳离子脂质、磷脂酰甘油类，诸如DOPG(二油酰磷脂酰甘油)、DPPG(二棕榈酰磷脂酰甘油)和二硬脂酰磷脂 酰甘油；磷脂酰丝氨酸(和它们的衍生物)；磷脂酰丝氨酸类，诸如二油酰基-或二棕榈酰磷脂酰丝氨酸；二季铵盐类诸如N,N'-二油烯基-N,N,N',N'-四甲基-1,2-乙二胺(TmedEce)、N,N'-二油烯基-N,N,N',N'-四甲基-1,3-丙二胺(PropEce)、N,N'-二油烯基-N,N,N',N'-四甲基-1,6-己二胺(HexEce)和它们的对应的N,N'-二鲸蜡基饱和类似物(TmedAce、PropAce和HexAce)、二磷脂酰甘油类；脂肪酸酯类；单阳离子转染脂质诸如1-脱氧-1-[双十六烷基(甲基)铵基]-D-木糖醇；1-脱氧-1-[甲基(双十四烷基)铵基]-D阿拉伯糖醇；1-脱氧-1-[双十六烷基(甲基)铵基]-D-阿拉伯糖醇；1-脱氧-1-[甲基(双十八烷基)铵基]-D阿拉伯糖醇、甘油酯类；鞘脂类；强心脂类(cardolipin)；脑苷脂类；和神经酰胺类；以及它们2种或多种的混合物。中性脂质也可选自以下可商购获得的阳离子脂质混合物，例如，
Figure PCTCN2019125165-appb-000015
(N,NI,NII,NIII-四甲基-N,NI,NII,NIII-四棕榈基精胺(TMTPS)和二油酰基磷脂酰乙醇胺(DOPE)的1:1.5(M/M)制剂)、
Figure PCTCN2019125165-appb-000016
GS2888
Figure PCTCN2019125165-appb-000017
Figure PCTCN2019125165-appb-000018
和
Figure PCTCN2019125165-appb-000019
LIPOFECTAMINE
Figure PCTCN2019125165-appb-000020
LIPOFECTAMINE
Figure PCTCN2019125165-appb-000021
Figure PCTCN2019125165-appb-000022
TFXN TM、TRANSFAST TM、
Figure PCTCN2019125165-appb-000023
Figure PCTCN2019125165-appb-000024
vectamidine(3-十四烷基氨基-N-叔丁基-N'-十四烷基丙脒(又名双C14-脒)、
Figure PCTCN2019125165-appb-000025
以及其它。上面列出的辅助脂质的任意组合或混合物也可预期用于本公开的转染复合物。下述专利文件、专利申请或参考文献通过引用整体并入本公开，特别是它们的关于转染剂的公开内容，所述转染剂含有可用于构成本公开的转染复合物的阳离子和中性辅助脂质：美国专利6,075,012、6,020,202、5,578,475、5,736,392、6,051,429、6,376,248、5,334,761、5,316,948、5,674,908、5,834,439、6,110,916、6,399,663、6,716,882、5,627,159；PCT/US/2004/000430，公开号为WO04063342A2；PCT/US/9926825，公开号为WO0027795A1；PCT/US/04016406，公开号为WO04105697；和PCT/US2006/019356，公开号为WO07130073A2。
本公开使用的术语“聚乙二醇化脂质”通常表示，与一种或多种聚乙二醇部分共价地缀合的脂质。用于本公开的转染复合物实施方案的聚乙二醇化脂质包括：基于磷脂酰乙醇胺(PE)的聚乙二醇化脂质，例如，1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-MW]，其中MW表示聚乙二醇部分的平均摩尔质量。这样的二肉豆蔻酰基-PEG-PE脂质被统称为14:0PEG(MW)PE。聚乙二醇部分的平均MW可以是例如25、350、550、750、1000、2000、3000、5000、6000、8000或12000。基于磷脂酰乙醇胺的聚乙二醇化脂质的脂肪酸链可以包括，例如，1,2-二油酰基(诸如就18:1PEG(MW)PE而言)、1,2-二棕榈酰基(诸如就16:0PEG(MW)PE而言)或1,2-二硬脂酰基(诸如就18:0PEG(MW)PE而言)。其它基于磷脂酰乙醇胺(PE)的聚乙二醇化脂质包括，例如，1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺-N-[MOD(聚乙二醇)-MW]，也被称作DSPE-MODPEG(MW)，其中MOD表示官能部分，诸如胺、生物素、羧酸、叶酸酯、马来酰亚胺、PDP或羧基荧光素部分。这些基于PE的聚乙二醇化脂质中，聚乙二醇部分的平均摩尔质量MW可以为例如2000或5000。用于本公开所述的实施方案的聚乙二醇化脂质也包括基于神经酰胺的聚乙二醇化脂质，例如，N-辛酰基-鞘氨醇-1-{琥珀酰[甲氧基(聚乙二醇)MW]}，也被称为C8PEG(MW)神经酰胺，其中MW是例如750、2000或5000。可替换地，所述脂肪酸部分可以具有N-棕榈酰(C16)基(诸如就C16PEG(MW)神经酰胺而言)。在一些实施方式中，本公开的转染复合物中使用的聚乙二醇化脂质为1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)-2000]。
本公开提供的转染复合物通常是胶体制剂，但是也可以是干燥的制剂。本公开提供的转染复合物是一种聚集体，它可以是单层和多层的脂质体，可以是囊泡、胶束和无定形的聚集体。
在一些实施方式中，本公开的转染复合物还包含生物活性剂。术语“生物活性剂”可以是组合物、复合物、化合物或分子，该生物活性剂具有生物学效应，或者修饰、造成、促进、增强、阻断或减少生物学效应，或者增强或限制具有生物学效应的第二分子的产生或活性、与所述第二分子反应和/或结合。所述第二分子可以、但不一定是内源分子(例如，通常存在于靶细胞中的分子，诸如蛋白或核酸)。生物学效应可以是，但不限于：刺激或造成免疫反应应答的效应；影响细胞、组织或生物体(例如，动物)中的生物学过程的效应；影响病原体或寄生物中的生物学过程的效应；产生可检测信号或造成可检测信号的产生的效应；调节蛋白或多肽的表达的效应；终止或抑制蛋白或多肽的表达的效应；或造成或增强蛋白或多肽的表达的效应。
在一些实施方式中，合适的生物活性剂可以包括这样的分子：所述分子能够与本公开所述的含胺转染试剂形成转染复合物，且当被递送至一个或多个细胞内部或被递送至体内或体外组织时会引起生物应答。用于本公开所述的实施方式中的生物活性剂可以是阳离子的、中性的或阴离子的试剂。在一些实施方式中，所述生物活性剂为小分子、功能性寡核苷酸或药物蛋白，或者它们中任意一种的药物前体或药学上可接受的盐。在本公开的上下文中，术语“药物前体”也称为“前药”或“前体药物”，是指药物经过化学结构修饰后得到的在体外无活性或活性较小、在体内经酶或非酶的转化释放出活性药物而发挥药效的化合物。
在一些实施方式中，生物活性剂可以选自、但不限于：核酸、多肽、抗体、寡肽、治疗性的肽或蛋白分子、肽核酸(PNA)、阳离子的、阴离子的或中性的有机分子或药物，或它们的药学上可接受的盐。
在一些实施方式中，所述生物活性剂为功能性寡核苷酸，或者它们的药物前体或药学上可接受的盐。所述功能性寡核苷酸能够通过与靶序列之间产生稳定且特异性的杂交，利用RNA激活(RNA activation，RNAa)、RNA干扰(RNA interference，RNAi)、反义核酸技术或外显子跳跃(exon skipping)技术原理，上调或下调靶基因的表达，或导致mRNA可变剪接。在一些方面，功能性寡核苷酸还可以是与靶蛋白之间产生稳定且特异性地结合的核酸结构，如适配体。此外，本领域技术人员容易理解的是，多核苷酸(例如mRNA本身或其片段)也同样适用于与本公开提供的含胺转染试剂形成转染复合物以实现向细胞的转染递送，从而调节mRNA转录出的蛋白质的表达。因此，在上下文中，“功能性寡核苷酸”的概念也可涵盖mRNA或其片段。
在一些实施方式中，所述功能性寡核苷酸能够与靶序列发生相互作用，从而影响靶序列分子的正常功能，如导致发生mRNA断裂或翻译阻遏或外显子跳跃引发mRNA可变剪接。在一些实施方式中，所述功能性寡核苷酸可以与靶序列的碱基基本上互补、实质上反向互补或者完全反向互补。在一些实施方式中，所述功能性寡核苷酸可以与靶序列80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上的碱基互补，或者与靶序列完全互补。在一些实施方式中，所述功能性寡核苷酸可以含有1个、2个或3个不与靶序列互补的碱基。在一些实施方式中，所述功能性寡核苷酸选自脱氧核糖核酸或核糖核酸、以及具有修饰的脱氧核糖核 酸或核糖核酸。在一些实施方式中，所述功能性寡核苷酸是单链或双链的。在一些实施方式中，所述功能性寡核苷酸可以是单链的DNA、RNA或DNA-RNA嵌合体(chimera)，或者是双链的DNA、RNA或DNA-RNA杂交体(hybrids)。
由此，在一些实施方式中，适合包含于本公开的转染复合物中的功能性寡核苷酸可以是小干扰RNA(siRNA)、微小RNA(microRNA)、抗微小RNA(antimiR)、微小RNA拮抗剂(antagomir)、微小RNA模拟物(microRNA mimics)、诱饵寡核苷酸(decoy)、免疫刺激物(immune stimulatory)、G-四极子(G-quadruplex)、可变剪接体(splice altering)、单链RNA(ssRNA)、反义核酸(antisense)、核酸适配体(Nucleic Acid Aptamer)、小激活RNA(small activating RNA，saRNA)、茎环RNA(stem-loop RNA)或DNA中的一种。在进一步的实施方式中，适合包含于本公开的转染复合物中的功能性寡核苷酸可以是WO2009082607A2、WO2009073809A2或WO2015006740A2中公开的寡核苷酸，以引用的方式将其整体内容并入本公开。在一些实施方式中，适合包含于本公开的转染复合物中的功能性寡核苷酸为双链寡核苷酸。在一些实施方式中，适合包含于本公开的转染复合物中的功能性寡核苷酸为siRNA。
生物活性剂是功能性寡核苷酸为例本公开的转染复合物可以将功能性寡核苷酸递送至细胞，调节这些细胞中特定基因的表达，这些细胞可以是各种细胞。在一些实施方式中，所述细胞是肝细胞。在一些实施方式中，所述特定基因可以是肝细胞中表达的内源性基因，也可以是在肝细胞中繁殖的病原体基因。在肝细胞中表达的基因可以是例如ApoB、ApoC3、ANGPTL3、PCSK9、SCD1、TIMP-1、Col1A1、FVII、STAT3、p53、HBV、HCV基因。在一些实施方式中，所述在肝细胞中表达的基因是HBV基因、ANGPTL3基因或APOC3基因。在本公开的上下文中，上述基因序列是公知的，例如，HBV基因是指序列如Genbank注册号NC_003977.1所示的基因；ANGPTL3基因是指mRNA序列如Genbank注册号NM_014495.3所示的基因；APOC3基因是指mRNA序列如Genbank注册号NM_000040.1所示的基因。
在一些实施方式中，“靶序列”是靶mRNA。在本公开的上下文中，“靶mRNA”是指在细胞中异常表达的基因对应的mRNA，它既可以是过量表达的基因对应的mRNA，或者是表达不足的基因对应的mRNA。由于大部分疾病源于mRNA的过量表达，因此，在本公开中，靶mRNA尤其指过量表达的基因对应的mRNA。在本公开的一些实施方式中，相应于上述异常表达的基因，所述靶mRNA可以是ApoB、ApoC3、ANGPTL3、PCSK9、SCD1、TIMP-1、Col1A1、FVII、STAT3、p53、HBV、HCV基因对应的mRNA。在一些实施方式中，所述靶mRNA可以是HBV基因对应的mRNA、或者ANGPTL3基因表达的mRNA、或者APOC3基因表达的mRNA。
在不愿受到限制的情况下，在下面的实施方式和实施例中，详细描述了本公开的转染复合物中的功能性寡核苷酸为小干扰RNA(siRNA)的情况。这并不代表本公开的转染复合物中的生物活性剂仅可以是siRNA。根据转染复合物的详细说明，可以设想其它生物活性剂、特别是其它功能性寡核苷酸与本公开提供的含胺转染试剂形成转染复合物时也会有类似的作用。
本领域技术人员公知，siRNA含有核苷酸基团作为基本结构单元，所述核苷酸基团含有磷酸基团、核糖基团和碱基。通常具有活性的，即功能性的siRNA的长度约为12-40个 核苷酸，在一些实施方式中约为15-30个核苷酸，所述siRNA中的每个核苷酸可以独立地是修饰或未修饰的核苷酸，为了增加稳定性，所述siRNA中至少一个核苷酸是修饰的核苷酸。
本公开的发明人发现，下面的实施方式中所述的siRNA具有较高的活性、较高的稳定性、较低的脱靶效应和/或较低的毒性，因而可以作为本公开中的生物活性剂。
在一些实施方式中，本公开的转染复合物中的siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，该siRNA含有正义链和反义链，其中，所述正义链包含核苷酸序列1，所述反义链包含核苷酸序列2，所述核苷酸序列1和所述核苷酸序列2的长度相等，并且均为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25个核苷酸，并且至少部分地反向互补形成互补双链区，所述核苷酸序列2的至少一部分与第一段核苷酸序列互补，所述第一段核苷酸序列为靶mRNA中的一段核苷酸序列。在一些实施方式中，所述核苷酸序列1和所述核苷酸序列2的长度均为19、20或21个核苷酸。在一些实施方式中，所述核苷酸序列1和所述核苷酸序列2的长度均为19个核苷酸。
在一些实施方式中，所述siRNA是指在3mg/kg的剂量(以siRNA计)下，能够抑制至少50％乙型肝炎病毒基因表达、至少50％血管生成素样蛋白3基因表达或者至少50％载脂蛋白C3基因表达的siRNA。在一些实施方式中，所述siRNA指在剂量为3mg/kg时，能够抑制至少55％、60％、65％、70％、75％或80％HBV基因、ANGPTL3基因或APOC3基因表达。
在一些实施方式中，所述核苷酸序列1与所述第一段核苷酸序列长度相等，且不超过3个核苷酸差异；所述核苷酸序列2与核苷酸序列B长度相等，且不超过3个核苷酸差异；所述核苷酸序列B为与所述第一段核苷酸序列完全反向互补的核苷酸序列。在不愿受到限制的情况下，这些特定的核苷酸差异并不会显著降低转染复合物的靶基因抑制能力，而这些包含特定核苷酸差异的转染复合物也在本公开的保护范围之内。
在一些实施方式中，所述核苷酸序列1和所述核苷酸序列2基本上反向互补、实质上反向互补或完全反向互补。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；完全互补指两段核苷酸序列之间不存在碱基错配。
在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在核苷酸差异，指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或者核苷酸类似物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
在一些实施方式中，所述核苷酸序列1与所述第一段核苷酸序列不多于1个核苷酸差异，和/或所述核苷酸序列2与所述核苷酸序列B不多于1个核苷酸差异。在一些实施方式中，所述核苷酸序列2与所述核苷酸序列B之间的核苷酸差异包括按照5'末端到3'末端的方向，所述核苷酸序列2上的第一个核苷酸Z'位置上的差异。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1上的最后一个核苷酸Z是与Z'互补的核苷酸。
在一些实施方式中，所述正义链还含有核苷酸序列3，所述反义链还含有核苷酸序列4， 所述核苷酸序列3和所述核苷酸序列4的长度相等且均为1-4个核苷酸，所述核苷酸序列3连接在所述核苷酸序列1的5'末端，并且所述核苷酸序列4连接在所述核苷酸序列2的3'末端，所述核苷酸序列4与第二段核苷酸序列互补，该第二段核苷酸序列是指靶mRNA中与所述第一段核苷酸序列相邻、且长度与所述核苷酸序列4相同的核苷酸序列。在一些实施方式中，所述核苷酸序列3和所述核苷酸序列4实质上反向互补或完全反向互补。因此，所述正义链和反义链的长度可以是19-23个核苷酸。
在一些实施方式中，所述siRNA还含有核苷酸序列5，所述核苷酸序列5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；在一些实施方式中，所述核苷酸序列5的长度为1或2个核苷酸。这样，在一些实施方式中，所述siRNA的正义链和反义链的长度之比可以是19/20、19/21、20/21、20/22、21/22、21/23、22/23、22/24、23/24或23/25。
在一个实施方式中，所述核苷酸序列5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5为连续的2个脱氧胸腺嘧啶核苷酸、连续的2个尿嘧啶核苷酸、或者与第三段核苷酸序列互补，所述第三段序列是指靶mRNA中与所述第一段核苷酸序列相邻、或者和所述第二段核苷酸序列相邻，并且长度与所述核苷酸序列5相等的核苷酸序列。在一个实施方式中，所述siRNA的正义链和反义链的长度之比为19/21或21/23，此时，所述siRNA具有更好的靶mRNA沉默活性。
在一些实施方式中，所述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，所述siRNA不含修饰的核苷酸基团；在一些实施方式中，所述siRNA含有修饰的核苷酸基团。
目前，本领域存在多种可用于修饰siRNA的方式，例如骨架修饰(也称为核苷酸间连接修饰，如磷酸基团修饰)、核糖基团修饰及碱基修饰(例如，请参见Watts，J.K.，G.F.Deleavey and M.J.Damha，Chemically modified siRNA:tools and applications.Drug Discov Today，2008.13(19-20)：p.842-55，以引用的方式将其整体内容并入本公开)。
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被修饰，比如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。
在本公开的一些实施方式中，所述正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基。换句话说，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基(或修饰的磷酸酯基和/或修饰的核糖基)。在本公开的一些实施方式中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。
在一些实施方式中，正义链和反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。
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，具有以下式(207)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。
核糖基2'位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2'-烷氧基修饰的核苷酸、2'-经取代的烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-脱氧核苷酸中的一种。
在一些实施方式中，2'-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2'-OMe)，如式(208)所示。2'-经取代的烷氧基修饰的核苷酸，例如可以是2'-O-甲氧基乙基修饰的核苷酸(2'-MOE)，如式(209)所示。在一些实施方式中，2'-氨基修饰的核苷酸(2'-NH 2)如式(210)所示。在一些实施方式中，2'-脱氧核苷酸(DNA)如式(211)所示。
Figure PCTCN2019125165-appb-000026
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的基团。在一些实施方式中，所述核苷酸类似物可以为如异核苷酸、桥联核酸(bridged nucleic acid，简称BNA)核苷酸或无环核苷酸。
BNA核苷酸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环的2'-、4'-位处以提供一个2',4'-BNA核苷酸，如LNA、ENA、cET BNA，其中，LNA如式(212)所示，ENA如式(213)所示，cET BNA如式(214)所示。
Figure PCTCN2019125165-appb-000027
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸，如解锁核酸(UNA)核苷酸或甘油核酸(GNA)核苷酸，其中，UNA如式(215)所示，GNA如式(216)所示。
Figure PCTCN2019125165-appb-000028
Figure PCTCN2019125165-appb-000029
其中，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，例如，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(217)或(218)所示。
Figure PCTCN2019125165-appb-000030
其中，Base表示碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，所述甲氧基修饰的核苷酸指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”、“2'-氟修饰的核苷酸”、“核糖基团的2'-羟基被氟取代的核苷酸”和“2'-氟代核糖基”意义相同，均指核苷酸的2'-羟基被氟取代而形成的具有如式(207)所示结构的化合物；“甲氧基修饰的核苷酸”、“2'-甲氧基修饰的核苷酸”、“核糖基团的2'-羟基被甲氧基取代的核苷酸”和“2'-甲氧基核糖基”意义相同，均指核苷酸核糖基团的2'-羟基被甲氧基取代，而形成如式(208)所示的结构。
在一些实施方式中，所述siRNA是具有以下修饰的siRNA：按照5'末端到3'末端的方向，所述siRNA的正义链中所述核苷酸序列1的第7、8、9位的核苷酸为-氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列2的第2、6、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在一些实施方式中，所述siRNA是具有以下修饰的siRNA：或者按照5'末端到3'末端的方向，所述siRNA的正义链中所述核苷酸序列1的第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列2的第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在一些实施方式中，所述siRNA是具有以下修饰的siRNA：按照5'末端到3'末端的方向，所述siRNA的正义链中所述核苷酸序列1的第7、8和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，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中所述核苷酸序列2的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。
在本公开所述siRNA的一些具体实施方式中，所述核苷酸含有磷酸基团修饰。在本公开的上下文中，磷酸基团修饰在一个实施方式中为如下式(201)所示的硫代磷酸(phosphorthioate)修饰，即，用一个硫原子取代磷酸二酯键中的非桥氧原子，从而以硫代磷酸二酯键替换磷酸二酯键。在一些实施方式中，该修饰能稳定siRNA的结构，保持碱基 配对的高特异性和高亲和力。
Figure PCTCN2019125165-appb-000031
根据本公开的一些实施方式，所述siRNA中，硫代磷酸酯基连接存在于由以下位置的组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯基连接存在于除正义链5'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于除正义链3'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于以下位置中的至少一处：
所述正义链的5'末端端部第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的5'末端端部第2个核苷酸和第3个核苷酸之间的连接；
所述正义链的3'末端端部第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的3'末端端部第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的5'末端端部第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的5'末端端部第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的3'末端端部第1个核苷酸和第2个核苷酸之间的连接；以及
所述反义链的3'末端端部第2个核苷酸和第3个核苷酸之间的连接。
按照本公开的一些实施方式，所述siRNA分子的反义链序列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。
在一些实施方式中，5'-磷酸核苷酸可具有式(202)所示结构：
Figure PCTCN2019125165-appb-000032
同时，常用的所述5'-磷酸类似物修饰的核苷酸的种类是本领域技术人员公知的，例如，Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48中公开的如下如式(203)-(206)所示的4种核苷酸：
Figure PCTCN2019125165-appb-000033
Figure PCTCN2019125165-appb-000034
其中，R表示选自于由H、OH、F和甲氧基所组成的组的基团；
Base表示选自A、U、C、G或T的碱基。
在一些实施方式中，5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸为式(203)所示的含有乙烯基磷酸酯(E-vinylphosphonate，E-VP)的核苷酸、式(202)所示的含有5'-磷酸修饰的核苷酸或式(205)所示的含有5'-硫代磷酸修饰的核苷酸。
表1A-1F中示出了可作为生物活性剂的siRNA，使用这些siRNA的本公开提供的转染复合物在保持较高活性的同时，同样具有出人意料的低毒性的特点。
表1A
Figure PCTCN2019125165-appb-000035
表1B
Figure PCTCN2019125165-appb-000036
Figure PCTCN2019125165-appb-000037
表1C
Figure PCTCN2019125165-appb-000038
Figure PCTCN2019125165-appb-000039
表1D
Figure PCTCN2019125165-appb-000040
表1E
Figure PCTCN2019125165-appb-000041
Figure PCTCN2019125165-appb-000042
表1F
Figure PCTCN2019125165-appb-000043
Figure PCTCN2019125165-appb-000044
*S：正义链；AS：反义链
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2'-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2'-氟修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间的连接为硫代磷酸酯基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸，在一些实施方式中为乙烯基磷酸酯修饰的核苷酸、5'-磷酸修饰的核苷酸或硫代磷酸酯修饰的核苷酸。
本领域技术人员清楚知晓的是，可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。
在上文及下文中，在描述本公开的功能性寡核苷酸的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)指，根据欲制备的RNA序列，在固相亚磷酰胺合成中使用的“未修饰或修饰RNA亚磷酰胺(unmodified or modified RNA phosphoramidite)”，固相亚磷酰胺合成是本领域合成RNA众所周知的方法。当所述RNA为siRNA时，所述方法 通常包括，按照欲制备的siRNA中正义链或反义链的核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，合成正义链和反义链，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应，得到连接在固相载体上的正义链和连接在固相载体上的反义链；脱除保护基并与固相载体切割，分别分离纯化，获得siRNA的正义链或反义链，退火。在本公开中，RNA亚磷酰胺同时也指核苷亚磷酰胺(nucleoside phosphoramidites)。除非另有说明，本公开所使用的核苷单体均可商购得到。
所述固相合成的其它条件，包括核苷单体脱保护条件，脱保护试剂种类和用量，偶联反应条件，偶联试剂的种类和用量，盖帽反应的条件，盖帽试剂的种类和用量，氧化反应条件，氧化试剂种类和用量，硫化反应条件，硫化试剂种类和用量采用本领域中常规使用的各种试剂、用量和条件。
例如，在一些实施方式中，所述固相亚磷酰胺合成可使用如下条件：
核苷单体脱保护条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为30-300秒，在一些实施方式中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方式中为二氯乙酸。脱保护试剂与固相载体上4,4'-二甲氧基三苯甲基保护基的的摩尔比可以为2:1-100:1，在一些实施方式中为3:1-50:1。
偶联反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，固相载体上连接的核酸序列与核苷单体的摩尔比可以为1:1-1:50，在一些实施方式中为1:5-1:15；固相载体上连接的核酸序列和偶联试剂的摩尔比为1:1-1:100，在一些实施方式中为1:50-1:80，反应时间可以为200-3000秒，例如为500-1500秒。偶联试剂选自1H-四氮唑、5-乙硫基-1H-四氮唑、5-苄硫基-1H-四氮唑中的一种或多种，例如为5-乙硫基-1H-四氮唑。
盖帽反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为5-500秒，在一些实施方式中为10-100秒，盖帽试剂的选择与前述相同。盖帽试剂的总量与固相载体上连接的核酸序列的摩尔比为1:100-100:1，在一些实施方式中为1:10-10:1。在盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑的情况下，乙酸酐、N-甲基咪唑以及固相载体上连接的核酸序列的摩尔比可为1:1:10-10:10:1，在一些实施方式中为1:1:2-2:2:1。
氧化反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为1-100秒，在一些实施方式中为5-50秒，氧化试剂在一些实施方式中为碘(在一些实施方式中，以碘水的形式提供)。氧化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比可以为1:1-100:1，在一些实施方式中为5:1-50:1。在一些实施方式中，所述氧化反应在四氢呋喃:水:吡啶＝3:1:1-1:1:3的混合溶剂中进行。
当siRNA中两个核苷酸之间的连接为硫代磷酸酯基连接时，以硫化代替氧化步骤。硫化反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为50-2000秒，在一些实施方式中为100-1000秒，硫化试剂在一些实施方式中为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比为10:1-1000:1，在一些实施方式中为10:1-500:1。在一些实施方式中，所述硫化反应在乙腈:吡啶＝1:3-3:1的混合溶剂中进行。
在将所有核苷单体连接之后，退火之前，该方法还包括分离出siRNA的正义链和反义链。分离的方法为本领域技术人员所公知，一般包括将合成得到的核苷酸序列从固相载体上切割下来，脱除碱基上和磷酸基上的保护基团，纯化和脱盐。
将合成得到的核苷酸序列从固相载体上切割下来，并脱除碱基上和磷酸基上的保护基团，可按照siRNA合成中常规的切割和脱保护方法进行。例如，将得到的连接有固相载体的核苷酸序列与浓氨水接触。其中，所述浓氨水可以是25-30重量％的氨水，浓氨水的用量与目标siRNA序列相比可以为0.2ml/μmol-0.8ml/μmol。
在所合成的核苷酸序列上存在至少一个2'-TBDMS保护时，所述方法还包括将脱除了固相载体的核苷酸序列与三乙胺三氢氟酸盐接触，以脱除该2'-TBDMS保护。此时，所得到的目标siRNA序列中的相应核苷酸具有游离的2'-羟基。三乙胺三氢氟酸盐纯品的用量与目标siRNA序列相比可以为0.4ml/μmol-1.0ml/μmol。这样即可得到可用于本公开的转染复合物的siRNA。
纯化和脱盐的方法是本领域技术人员熟知的。例如，可利用制备型离子色谱纯化柱，通过NaBr或NaCl的梯度洗脱，完成核酸的纯化；产品收集合并后，可采用反相色谱纯化柱进行脱盐。
这样得到的siRNA中，核苷酸之间的磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子基本与钠离子结合，所得到的siRNA基本以钠盐形式存在。可以采用熟知的离子交换方法，用氢离子和/或其他阳离子(例如，其它碱金属离子或铵离子)取代所述钠离子，得到其他形式的siRNA。
在合成过程中，可随时对核酸序列的纯度和分子量进行检测，更好地把控合成质量，此类检测的方法为本领域技术人员所公知。例如，可通过离子交换色谱检测核酸纯度，并通过液质联用色谱(LC-MS)测定分子量。
退火的方法也是本领域技术人员熟知的。例如，可简单地将所合成的正义链(S链)与反义链(AS链)以等摩尔比混合在注射用水中加热至70-95℃，随后室温冷却，使其通过氢键形成双链结构。这样即可得到可用于本公开的转染复合物的siRNA。
在获得所述siRNA后，在一些实施方式中，还可利用例如液质联用色谱方法，通过例如分子量检测的方式对所合成siRNA进行表征，确定所合成的siRNA的序列为期望的siRNA的序列，例如为表1中所列的序列之一。
在本公开提供的转染复合物中，可以没有生物活性剂，所述生物活性剂的含量可以在较大范围内变动，只要对受试者没有危害即可。在一些实施方式中，本公开的转染复合物中的生物活性分子与总脂质的重量比在约1:1-1:200的范围内，其中所述总脂质为所述关键脂质(即，本公开的含胺转染试剂)、辅助脂质和聚乙二醇化脂质的总和。在一些实施方式中，所述重量比在从约1:1至约1:50、从约1:1至约1:30、从约1:1至约1:20、从约1:2至约1:18、从约1:3至约1:17、从约1:4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，生物活性剂与脂质总量的重量比为约1:3、1:4、1:5、1:6、1:7、1:8、1:9、或1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17或1:18。
在某些非限制性的实施方案中，由所述siRNA与上述含胺转染试剂形成的转染复合物颗粒具有约30nm至约200nm的平均直径(即，表观平均粒径)，通常为约40nm至约135nm，在一些实施方式中，该转染复合物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，该转染复合物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，本公开的转染复合物颗粒具有均一的分散性(PDI)。在一些实施 方式中，本公开的转染复合物颗粒具有0.03-0.5的分散性。在一些实施方式中，本公开的转染复合物颗粒具有0.05-0.35的分散性。在一些实施方式中，本公开的转染复合物颗粒具有0.07-0.166的分散性。
在一些实施方式中，本公开的转染复合物具有70.3％-99.9％的包封率。在一些实施方式中，本公开的转染复合物具有75.0％-99.0％的包封率。在一些实施方式中，本公开的转染复合物具有79.3％-98.15％的包封率。
在一些实施方式中，本公开的转染复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。
本公开提供的转染复合物的制备方法包括提供一种含有关键脂质的溶液，将所述含有关键脂质的溶液孵育，所述关键脂质为本公开提供的含胺转染试剂。
在一些实施方式中，所述含有关键脂质的溶液还含有辅助脂质和聚乙二醇化脂质。关键脂质与辅助脂质和聚乙二醇化脂质的比例如前所述。
在一些实施方式中，该方法还包括将孵育后的脂质体制剂浓缩或稀释，在一些实施方式中，该方法还包括去除杂质和/或除菌。
在一些实施方式中，该方法还包括在孵育之前，将含有关键脂质的溶液与含有生物活性剂的溶液混合。所述生物活性剂的种类和含量如前所述。
含有关键脂质的溶液和含有生物活性剂的溶液中各自溶剂及其用量均为常规的选择。孵育的条件可采用常规的条件。
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，除菌的方法可以采用常规的方法。例如，去除杂质的方法可以采用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。可以在0.22μm滤器上过滤除菌。
在一个具体的实施方式中，以生物活性剂为siRNA为例，本公开提供的转染复合物可以按照如下方法制备：
将本公开提供的含胺转染试剂(关键脂质)、辅助脂质和聚乙二醇化脂质按照上述摩尔比在醇中混匀得到含有关键脂质的溶液；醇的用量使得到的含有关键脂质的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，在一些实施方式中，所述醇选自乙醇、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。
将含有关键脂质的溶液和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。含有关键脂质的溶液和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，得到本公开提供的转染复合物，所述转染复合物的理化参数为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％，表观平均粒径(平均颗粒直径) 为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
本公开的转染复合物的应用
在一些实施方式中，提供了本公开的转染复合物在制备用于治疗和/或预防由细胞中特定基因的表达而引起的病理状况或疾病的药物中的用途。
在一些实施方式中，本公开提供了一种治疗和/或预防由细胞中特定基因的表达而引起的病理状况或疾病的方法，该方法包括将有效量的本公开的包含生物活性剂的转染复合物给予有需要的受试者。
以所包含的生物活性剂为siRNA为例，通过将本公开的转染复合物给予有需要的受试者，可以通过RNA干扰的机制达到治疗和/或预防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，本公开的转染复合物可用于治疗和/或预防由细胞中特定基因的表达而引起的病理状况或疾病，或用于制备用于治疗和/或预防由细胞中特定基因的表达而引起的病理状况或疾病的药物。
所述特定基因可以是细胞中表达的内源性基因，也可以是在细胞中繁殖的病原体基因。在一些实施方式中，所述特定基因选自ApoB、ApoC3、ANGPTL3、PCSK9、SCD1、TIMP-1、Col1A1、FVII、STAT3、p53、HBV、HCV基因。相应地，生物活性剂选自能够特异性地与上述基因对应的mRNA结合的生物活性剂，如靶向上述基因对应的mRNA的siRNA。在一些实施方式中，所述特定基因选自乙型肝炎病毒基因、血管生成素样蛋白3基因或者载脂蛋白C3基因。相应地，所述疾病选自慢性肝病、肝炎、肝纤维化疾病、肝增生性疾病和血脂异常。在一些实施方式中，所述血脂异常为高胆固醇血症、高甘油三酯血症或动脉粥样硬化。在一些实施方式中，本公开提供的转染复合物也可用于治疗其它疾病，包括以不需要的细胞增殖为特征的疾病、血液疾病、代谢疾病和以炎症为特征的疾病。增殖疾病可以是良性或恶性疾病，例如癌症或细胞瘤。血液学或炎症疾病可以是涉及凝血因子、补体介导的炎症或纤维化的疾病。代谢疾病包括血脂异常和葡萄糖调节的不规则性。在一些实施方案中，通过施用一种或多种具有与参与细胞的基因序列高度同源的寡核苷酸来治疗疾病。部分可选择的生物活性剂在上文中已经进行了详细说明。
本公开所使用的术语“给药/给予”是指通过使得至少部分地将本公开的转染复合物定位于期望的位点以产生期望效果的方法或途径，将本公开的转染复合物放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者体循环相比将更多转染复合物递送至特定位点；而全身给药导致将本公开的转染复合物递送至受试者的体循环。
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。
本公开所述的转染复合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD 50(使50％的群体死亡的致死剂量)和ED 50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指能引起50％实验对象出现阳性反应 时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开所述的转染复合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的BALB/c小鼠或CD1小鼠，对于包含siRNA的本公开的转染复合物，其siRNA用量可以为0.001-50mg/kg体重，在一些实施方式中为0.01-10mg/kg体重，在一些实施方式中为0.05-5mg/kg体重，在一些实施方式中为0.1-3mg/kg体重。
在一些实施方式中，本公开提供了一种抑制细胞中特定基因表达的方法，该方法包括将有效量的本公开的转染复合物与所述细胞接触，将本公开的转染复合物导入所述细胞，促使所述转染复合物中的生物活性剂进入细胞，起到抑制特定基因表达的目的。在一些实施方式中，所述细胞是肝细胞。在一些实施方式中，所述肝细胞可以选自SMMC-7721、HepG2、Huh7肝癌细胞系或分离的肝原代细胞。在一些实施方式中，所述细胞为分离的肝原代细胞。
采用本公开提供的方法抑制特定基因在细胞中表达，所提供的转染复合物中的生物活性剂、尤其是功能性寡核苷酸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，所述功能性寡核苷酸是siRNA，所提供的包含siRNA的转染复合物中的siRNA用量一般是这样的量：所述siRNA的量足以减少靶基因的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或0.05nM至约5nM的细胞外浓度。达到该局部浓度所需的量将随各种因素而变化，所述因素包括例如递送方法、递送部位、在递送部位和靶细胞或组织之间的细胞层的数目、递送途径(局部还是全身)。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。
有益效果
在一些实施方式中，与现有技术中描述的转染复合物相比，本公开提供的转染复合物在体内具有出人意料的低毒性和更高或至少相当的活性。
根据本公开的一些实施方式，本公开提供的包含siRNA的转染复合物显示出优异的靶基因抑制效果。例如，根据本公开的一个实施方式，本公开提供的包含针对ApoB基因表达的siRNA的转染复合物表现出优异的抑制ApoB基因表达的特性：在具有低脱靶效应的同时，能够在1mg/kg的剂量下抑制BALB/c小鼠肝脏中93.89％-94.30％的ApoB基因表达。同时，本公开提供的包含siRNA的转染复合物还能够有效地降低BALB/c小鼠中的血脂水平，在1mg/kg的剂量下可以接近以现有技术的转染复合物转染情况下的血清CHO和TG抑制水平，甚至能够达到更高的抑制水平。同时，与现有技术提供的相比，本公开提供的包含siRNA的转染复合物还显示出人意料的低肝脏急毒性，特别是与现有技术的转染复合物相比，在大体解剖中观察到的脏器病变发生在更高的浓度下，并且最大耐受剂量(MTD)提高至现有技术的转染复合物的至少1.4倍、甚至2.9倍以上。同时，在给予现有技术转染复合物的实验动物已经显示出明显的毒性病变现象，如肝脏变色、腹水时，给予同等剂量的本公开转染复合物的实验动物的大体解剖出人意料地未显示出明显异常。因此，本公开的含胺转染试剂以及转染复合物在生物活性剂、特别是功能性寡核苷酸的体内转染递送方面显示出卓越的潜力。
试剂盒
本公开提供了一种试剂盒，所述试剂盒包含有效量的本公开的转染复合物。
在一些实施方式中，本公开所述的试剂盒可在一个容器中提供转染复合物。在一些实施方式中，本公开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。在一些实施方式中，本公开所述的试剂盒可在不同于提供本公开所述生物活性剂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，所述试剂盒可在不同容器中提供所述转染复合物的各个或多个组分。在一些实施方式中，所述试剂盒可包含用于将所述转染复合物的各个或多个组分进行混合以获得本公开的转染复合物的说明书。
在本公开的试剂盒中，所述转染复合物或其组分可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述转染复合物或其组分基本上纯净和/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
以下将通过实施例对本公开进行详细描述。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，所用到的实验操作如核酸电泳、real-time PCR均参照Molecular Cloning(Cold Spring Harbor LBboratory Press(1989))所记载的方法进行。
若无其它说明，所使用的动物模型如下：
CD1小鼠：购于北京维通利华实验动物技术有限公司；
BALB/c小鼠：6-8周龄，购于北京维通利华实验动物技术有限公司。
实施例1含胺转染试剂及其制备
Figure PCTCN2019125165-appb-000045
(1-1)式N5_1化合物的制备
向10g 1,4-丁二胺(113.4mmol，1.0eq)中，加入500ml无水乙醇。在50℃下加热至固体全部溶解，加入52.9g 1,2-环氧十四烷(245.4mmol，2.2eq)，于50℃继续反应5h。将反应液冷却至室温，抽滤至干，滤饼用乙醇洗涤两次，每次10ml乙醇，干燥得式N5_1化合物(27g)，所得产品具有式(303)所示的结构，其中Y 1为亚丁基，R 2为2-羟基十四烷基。质谱检测结果：([M+H] +)：理论：513.53；实际：513.47。
(1-2)式N5_2化合物的制备
向步骤(1-1)获得的11g N5_1(21.4mmol,1.0eq)中加入12.2g N-(2,3-环氧丙基)邻 苯二甲酰胺(60.0mmol,2.8eq)，4.1g二异丙基乙胺(DIEA,32.1mmol,1.5eq)以及150ml DMF。在120℃反应6h，继续反应过夜。将反应液冷却至室温，并倾入1L水中，弃去水层，向残余物中加入50ml水和30ml二氯甲烷(DCM)，分出有机层，水层继续用20ml DCM萃取一次。合并有机层，蒸发溶剂至干，抽滤，干燥得式N5_2化合物(18g，91.8％)，所得产品具有式(302)所示的结构，其中，同一氮原子上的两个R 304共同组成邻苯二甲酰基保护基团，Y 2为2-羟基亚丙基。质谱检测结果：([M+H] +)：理论：919.64；实际：919.59。粗品未经进一步纯化直接投入下步反应。
(1-3)式N5化合物的制备
向步骤(1-2)获得的18g N5_2(19.6mmol.1.0eq)中加入6.9g 85wt％的水合肼(117.6mmol,6.0eq)以及400ml无水乙醇。加热回流2h。将反应液冷却至0℃，抽滤，滤饼用乙醇洗涤，合并母液，浓缩并抽滤至干，得粗品N5(13g)所得产品具有式(301)所示的结构，其中Y 2为2-羟基亚丙基。质谱检测结果：([M+H]+)：理论：659.63；实际：659.61。粗品未经进一步纯化直接投入下步反应。
(1-4)含胺转染试剂及其制备
Figure PCTCN2019125165-appb-000046
向步骤(1-3)获得的1.4g N5(2.12mmol,1.0eq)中加入1.23g DIEA(9.54mmol,4.5 eq)以及15ml DMF，在得到的溶液中加入2.1g化合物C1(4-溴巴豆酸十四醇酯，5.52mmol,2.6eq)，在50℃下反应2h。将反应液冷却至室温，并倒入1L水中，弃去水层，残余物加入50ml水中，用30ml DCM搅拌溶解，分出有机层，水层继续用20ml DCM萃取两次，合并有机层，干燥，过滤，蒸发溶剂至干。将产物用硅胶柱层析洗脱(洗脱剂：DCM:MeOH＝50:1-10:1(v/v))，以多个经标记的样品管按顺序依次收集洗脱出的层析级分，以TLC实时测定洗脱液是否具有单一组成，并将具有相同单一组成的层析级分分别合并，获得三个各自具有单一组成的样品组分，浓缩去除洗脱剂，得到含胺转染试剂1(280mg，纯度92.0％)、含胺转染试剂2(300mg，纯度91.7％)和含胺转染试剂3(110mg，纯度85.6％)，其中，以HPLC检测纯度，通过质谱检测分子量，确认含胺转染试剂1、含胺转染试剂2和含胺转染试剂3分别是具有式(101)、(102)、或(103)所示结构的含胺转染试剂。
实施例2转染复合物的制备
1)，生物活性剂siRNA及其合成
分别合成了如表2-1所示的siRNA1和siRNA2。
表2-1 siRNA的序列
Figure PCTCN2019125165-appb-000047
*SS：正义链；AS：反义链
注：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2'-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2'-氟修饰的核苷酸；字母组合dT表示胸腺嘧啶脱氧核苷酸。
除了上述序列不同外，siRNA1和siRNA2的合成条件完全相同。具体合成方法如下：
通过亚磷酰胺核酸固相合成的方法，利用通用固相载体(
Figure PCTCN2019125165-appb-000048
UnyLinker TM300 Oligonucleotide Synthesis Support，Kinovate Life Sciences公司，结构如式B80所示)起始循环合成正义链：
Figure PCTCN2019125165-appb-000049
按照上述序列顺序按照3'-5'的方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化四步反应。合成条件给定如下：
核苷单体以0.1M浓度的乙腈溶液提供，每一步的脱保护反应的条件相同，即温度为 25℃，反应时间为70秒，脱保护试剂为二氯乙酸的二氯甲烷溶液(3％v/v)，二氯乙酸与固相载体上4,4'-二甲氧基三苯甲基保护基的摩尔比为5:1。
每一步偶联反应条件均相同，包括温度为25℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1:10，固相载体上连接的核酸序列和偶联试剂的摩尔比为1:65，反应时间为600秒，偶联试剂为5-乙硫基-1H-四氮唑的0.5M乙腈溶液。
每一步盖帽条件均相同，包括温度为25℃，反应时间为15秒。盖帽试剂溶液为摩尔比为1:1的Cap1和Cap2的混合溶液，其中，Cap1和Cap2为盖帽试剂溶液，Cap1为20％(v/v)N-甲基咪唑的吡啶/乙腈混合溶液，吡啶与乙腈的体积比为3:5；Cap2为20％(v/v)乙酸酐的乙腈溶液；盖帽试剂与固相载体上连接的核酸序列的摩尔比为乙酸酐:N-甲基咪唑:固相载体上连接的核酸序列＝1:1:1。
每一步氧化反应条件相同，包括温度为25℃，反应时间为15秒，氧化试剂为浓度为0.05M的碘水。碘与偶联步骤中固相载体上连接的核酸序列的摩尔比为30:1。反应在四氢呋喃:水:吡啶＝3:1:1的混合溶剂中进行。
切割和脱保护条件如下：将合成的连接有载体的核苷酸序列加入浓度为25wt％的氨水中，氨水用量为0.5ml/μmol，在55℃反应16h，除去液体，真空浓缩至干。在氨水处理后，相对于单链核酸的量，用0.4ml/μmol N-甲基吡咯烷酮溶解产品，随后加入0.3ml/μmol三乙胺和0.6ml/μmol三乙胺三氢氟酸盐，脱除核糖上的2'-TBDMS保护。纯化与脱盐：利用制备型离子色谱纯化柱(Source 15Q)，通过NaCl的梯度洗脱，完成核酸的纯化。具体而言为：洗脱剂A：20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9:1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9:1(体积比)；洗脱梯度：洗脱剂A:洗脱剂B＝100:0-50:50梯度洗脱。收集产品洗脱液后合并，采用反相色谱纯化柱进行脱盐，具体条件包括采用葡聚糖凝胶柱进行脱盐，填料为葡聚糖凝胶G25，以去离子水洗脱。
检测：使用离子交换色谱(IEX-HPLC)进行检测；采用液质联用(LC-MS)分析分子量。
从而，该步骤中得到siRNA正义链SS。
利用通用固相载体(UnyLinker TM loaded
Figure PCTCN2019125165-appb-000050
Solid Supports，Kinovate Life Sciences公司)，合成siRNA的反义链AS。固相合成方法中的脱保护、偶联、盖帽、氧化反应条件，脱保护和切割，分离条件与合成正义链相同，得到siRNA反义链AS。
检测：纯度采用离子交换色谱(IEX-HPLC)进行检测；分子量采用液质联用(LC-MS)进行分析。
将SS链和AS链分别溶于注射用水中，得到40mg/ml的溶液。它们以等摩尔比混合，在50℃下加热15分钟，室温冷却后，得到siRNA1和siRNA2。
用超纯水(Milli-Q超纯水仪自制，电阻率18.2MΩ*cm(25℃))将siRNA稀释至0.2mg/mL的浓度。利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测并确认所获得的siRNA1和siRNA2具有表2-2中所列的核酸序列。
2)含siRNA1转染复合物及其制备
A)含胺转染试剂溶液的制备分别将上述得到的含胺转染试剂1-3和对比含胺转染试剂LC8中的一种、胆固醇、聚乙二醇化脂质(1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺-N-[甲 氧基(聚乙二醇)-2000(DPPE-PEG))以52:20:28的摩尔比与乙醇混合，得到分别包含含胺转染试剂1-3和LC8的4种含胺转染试剂乙醇溶液，4种含胺转染试剂乙醇溶液中含胺转染试剂的浓度均为6.50μM。
其中，对比含胺转染试剂LC8是按照中国专利CN 103380113B中实施例12制备的阳离子脂质87，该含胺转染试剂具有如式(901)所示的结构：
Figure PCTCN2019125165-appb-000051
B)siRNA溶液的制备
将siRNA1溶解于200mM醋酸钠(pH 5.2)溶液中，使siRNA1的浓度为0.4mg/ml，获得siRNA1醋酸钠水溶液。
C)转染复合物的制备
将A)得到4种含胺转染试剂乙醇溶液分别与B)得到的siRNA1醋酸钠水溶液在搅拌下混合，含胺转染试剂乙醇溶液的流速为1ml/20s、siRNA1醋酸钠水溶液的流速为4ml/20s搅拌速度为1000-1500转/分钟(rpm)将得到的产物分别在50℃的水浴锅中孵育10分钟。孵育后，在切相流系统，使用中空纤维柱100K Da进行超滤，超滤交换溶液为pH 7.4的PBS。超滤后的产物在0.22μm滤器上过滤除菌。分别得到转染复合物1-3和对比转染复合物1的水溶液。
3)含siRNA2转染复合物及其制备
按照与2)中A)同样的方法制备含胺转染试剂溶液，不同的是，所用含胺转染试剂分别为含胺转染试剂1-2和对比含胺转染试剂LC8，得到分别包含含胺转染试剂1-2和LC8的3种含胺转染试剂乙醇溶液。
按照与2)中B)同样的方法制备siRNA溶液的，不同的是用siRNA2代替siRNA1，得到siRNA2醋酸钠水溶液。
按照与2)中C)同样的方法制备转染复合物，不同是用上述得到的分别包含含胺转染试剂1-2和LC8的3种含胺转染试剂乙醇溶液代替所述4种含胺转染试剂乙醇溶液，用siRNA2醋酸钠水溶液代替siRNA1醋酸钠水溶液，得到转染复合物4、转染复合物5和对比转染复合物2。
测定转染复合物1-5和对比转染复合物1-2的包封率、表观平均粒径(颗粒平均直径)、分散性和siRNA浓度，检测结果见表2-2。
表2-2
Figure PCTCN2019125165-appb-000052
其中，包封率采用RiboGreen法检测，所用试剂(Quant-iT TMRNA Reagentand Kit)购于Thermo Fisher(Invitrogen)公司，货号R11490。根据说明书操作步骤检测样品中siRNA的荧光强度，再按照文献(J.Heyes et.al,Journal of Controlled Release,107(2005):276–287)所述方法计算包封率：
包封率＝[(Triton处理组荧光强度-无Triton处理组荧光强度)/Triton处理组荧光强度]×100％
以光散射法(ZetaPALS,Brookhaven Instruments)按照仪器说明书对表观平均粒径和分散性进行检测，并通过NANO DROP 2000(Thermo)测定siRNA浓度。
按表2-2中siRNA浓度和转染复合物组成计算可得，转染复合物1-5和对比转染复合物1-2中siRNA与总脂质的重量比如表2-3所示：
表2-3
Figure PCTCN2019125165-appb-000053
实施例3转染复合物对ApoB mRNA表达的抑制以及血脂影响
(1)小鼠给药方法
将6-8周龄，体重为18-25g的雌性BALB/c小鼠随机分成13组，分别记为阴性对照组 (1组，给予1×PBS溶液)、阳性对照组(3组，分别给予不同剂量的对比转染复合物1)、以及实验组(3×3组，分别给予不同剂量的转染复合物1、转染复合物2或转染复合物3)，其中阴性对照组6只小鼠，阳性对照组以及实验组每组各5只小鼠。给药方式均为单次尾静脉给药，给药剂量(以siRNA计)分别为1mg/kg、0.5mg/kg、0.1mg/kg，给药体积为10mL/kg。给药后24h进行眼眶采血，随后将动物处死，对动物进行大体解剖并观察脏器，收集肝脏组织，并分别用RNA later(Sigma Aldrich公司)保存待测；
(2)小鼠肝脏组织ApoB mRNA表达水平检测
对于前述阳性对照组和实验组中的每一组，分别向待测肝组织中加入1ml/g RNAVzol，加入钢珠在Tissuelyset II型全自动组织匀浆器中匀浆1分钟；向匀浆后的样品中加入0.2ml氯仿，振荡15s，静置3分钟；在4℃下以12000rpm离心15分钟，收取上清液；在上清液中加入0.5mL异丙醇，混匀，在室温下静置10分钟；在4℃下以12000rpm离心10分钟，弃掉上清液；加入1mL 75％酒精洗涤沉淀，在4℃下以12000rpm离心5分钟，弃掉上清液；晾干，加入150μL DEPC水溶解。获得提取的小鼠肝脏组织总RNA。
使用NANO DROP 2000(Thermo)测定所获得的提取的总RNA浓度，并进行1％琼脂糖凝胶电泳(Agarose，OXOID Lot：1315449)，以检测提取的总RNA质量。
RNA电泳条件：1％凝胶、0.8μg上样，Marker：1Kb DNA ladder(TRANS，Lot#I11112，Code:#BM201)，90V，20分钟。
采用实时荧光定量PCR检测肝组织中ApoB mRNA的表达水平，具体地：使用ImProm-IITM反转录试剂盒(Promega公司)按其说明书将提取的总RNA逆转录为cDNA，接着用荧光定量PCR试剂盒(北京康为世纪生物科技有限公司)分别检测肝组织中ApoB和GAPDH的mRNA的表达量，计算siRNA对肝组织中的ApoB mRNA表达的抑制效率。在该荧光定量PCR法中，以GAPDH基因作为内参基因，使用针对ApoB的引物和GAPDH的引物分别对ApoB和GAPDH的mRNA表达量进行检测。
表3引物及碱基序列
Figure PCTCN2019125165-appb-000054
PCR反应后，对产物进行1％琼脂糖凝胶电泳(Agarose，OXOID Lot：1315449)，以检测扩增质量。电泳条件：1％凝胶、6μL上样，Marker：100Kb DNA ladder(TRANS，Lot#K21022，Code:#BM301)，120V，25分钟。
其中，ApoB mRNA表达量＝(测试组ApoB mRNA的表达量/测试组GAPDH mRNA的表达量)/(对照组ApoB mRNA的表达量/对照组GAPDH mRNA的表达量)×100％，
抑制率＝[1-(测试组ApoB mRNA的表达量/测试组GAPDH mRNA的表达量)/(对照组ApoB mRNA的表达量/对照组GAPDH mRNA的表达量)]×100％，其中，抑制率指转染复合 物对ApoB mRNA表达量的抑制率
不同剂量本公开化合物制备的转染复合物和对比转染复合物在BALB/c小鼠体内对肝组织中ApoB mRNA表达水平的抑制率如图1和图2所示。
由图1和图2的结果可以看出，在小鼠中，转染复合物1和2的ApoB mRNA抑制活性接近甚至优于同等剂量下的对比转染复合物1。在低给药剂量(0.1mg/kg)下，与对比转染复合物1相比，转染复合物2显示出出乎意料的更高的ApoB mRNA抑制率。转染复合物2ApoB mRNA抑制率是对比转染复合物1的126.3％。
(3)血脂浓度检测
对于上述给予对比转染复合物1和转染复合物1-3的实验对象中，1mg/kg剂量组的实验动物以及给予1×PBS的阴性对照组的实验动物，将前述眼眶采集的血液按照组别分别进行离心得到血清，进一步使用PM1P000/3全自动血清生化仪(SABA，意大利)检测血清中总胆固醇(CHO)和甘油三酯(TG)的含量。检测结果示于图3和图4中。
由图3和图4的结果可见，以转染复合物1-3治疗后的小鼠血清中的CHO和TG的含量明显下降，并且显示出与同等剂量下对比转染复合物1至少相接近的血脂降低效果。特别地，以转染复合物1治疗的小鼠的总胆固醇浓度出人意料地显著低于对比转染复合物1治疗的小鼠，显示出明显更高的血脂降低效果。
实施例4转染复合物4-5和对比转染复合物2在CD1小鼠体内的急毒性测试
本实施例用于检测实施例2中所得的转染复合物4-5和对比转染复合物2在CD1小鼠体内的毒性反应情况。
将6-8周龄，体重为18-25g的CD1小鼠随机分组，分别记为阳性对照组(对比转染复合物2)和实验组(转染复合物4和转染复合物5)，每组6只小鼠(雌雄各3只，分别标记为M和F)。按表4所列剂量值分别向各组小鼠给予对比转染复合物2、转染复合物4或转染复合物5。
所有动物根据体重计算药量，采用尾静脉注射方式单次给药。给药后24h内观察毒性反应情况，并连续观察96h，记录毒性和死亡情况并进行剖检。结果参见表4所示。
表4转染复合物毒性测试
Figure PCTCN2019125165-appb-000055
Figure PCTCN2019125165-appb-000056
其中，“死亡情况汇总”一栏中，M和F分别表示小鼠的性别，M和F前的数字表示死亡的个数，例如，“1F”表示死亡一只雌性小鼠、“3M,3F”表示三只雄性小鼠和三只雌性小鼠全部死亡。“剖检情况”一栏中，带有斜杠“/”的数字组表示存活和肝局部变色的小鼠数量，斜杠“/”后的数字表示本组受试小鼠中存活小鼠数量，斜杠“/”前的数字表示本组存活的小鼠中发生肝脏局部变色的小鼠的数量，前面的文字说明肝脏变色的程度，例如“肝脏局部变色2/6”表明存活到最后的6只小鼠中，有2只发生了肝脏局部变色。
从表4的结果可见：
对于给予对比转染复合物2的实验动物组，在9.5mg/kg剂量组中未出现动物死亡，而且已经观察到明显的毒性反应但无严重的脏器病变；从首次出现动物死亡的10.8mg/kg剂量组之后的剂量组都出现动物死亡，在30.4mg/kg剂量组中动物全部死亡，并且其它剂量组中存活的动物剖检时发现不同程度的肝脏变色以及腹水病变。
对于给予转染复合物4的实验动物组，在15.2mg/kg剂量组中首次出现动物死亡；与同剂量的给予对比转染复合物2的实验动物组相比，直至13.0mg/kg剂量组中肝脏均未显示明显异常。
对于给予转染复合物5的实验动物组，在27.4mg/kg剂量组才首次出现动物死亡；与同剂量的给予对比转染复合物2的实验动物组相比，直至15.2mg/kg剂量组中肝脏均未显示明显异常，并且尤其是与对比转染复合物2的实验动物组相比，在30.4mg/kg剂量组中出人意料地没有任何动物死亡。
因此，对各转染复合物的最大耐受剂量(Maximum Tolerated Dose，MTD)大致估算如下，
对比转染复合物2：9.5-10.8mg/kg；
转染复合物4：13.7-15.2mg/kg；
转染复合物5：15.2-27.4mg/kg。
可见，与对比转染复合物相比，本公开的转染复合物在同等剂量下显示出更低的急毒性，并且最大耐受剂量方面，本公开的转染复合物出人意料地显示出明显更高的耐受值，特别是转染复合物5显示出与对比转染复合物2相比约1.4-2.9倍的最大耐受剂量值。另一方面，在10.8-15.2mg/kg体重的剂量范围内，对比转染复合物已经显示出高比例的肝毒性反应，而同等剂量下，给予本公开的转染复合物的实验动物在肝脏剖检中出人意料地显示出更低的毒性反应，特别是给予同等剂量范围的转染复合物5的实验动物在肝脏剖检中未 显示出任何明显异常。
根据以上实施例的结果可以发现，本公开的含胺转染试剂在递送效率与现有技术化合物相比相接近甚至更优的情况下，还出人意料地显示出显著更低的急毒性，因此在生物活性剂递送、特别是功能性寡核苷酸递送方面存在广泛而有效的应用前景。
以上详细描述了本公开的具体实施方式，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，在上述具体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，只要这种组合不违背本公开的思想，就同样应当视为本公开所公开的内容。

Claims (18)

  1. 一种含胺转染试剂，该含胺转染试剂为式(I)所示化合物或其药学上可接受的盐，
    Figure PCTCN2019125165-appb-100001
    其中，
    Y 1选自C2-C10的亚烷基或具有取代基的C2-C10亚烷基；
    每个Y 2相同或不同，独立地选自C2-C6的亚烷基或具有取代基的C2-C6亚烷基；
    每个R 1相同或不同，独立地选自H或式(I-i)所示的基团，每个R 2独立地为式(I-ii)所示的基团，
    Figure PCTCN2019125165-appb-100002
    其中，每个R a和每个R b各自独立地选自C6-C20直链烷基，每个R c各自独立地选自非胺类亲水性基团中的一种，
    Figure PCTCN2019125165-appb-100003
    表示基团共价连接的位点。
  2. 根据权利要求1所述的含胺转染试剂，其中，两个R 1均为H；或者，一个R 1为H，另一个R 1为(I-i)所示的基团。
  3. 根据权利要求1或2所述的含胺转染试剂，其中，每个R c各自独立地选自羟基、巯基、羧基、磷酸基团或聚乙二醇基团。
  4. 根据权利要求3所述的含胺转染试剂，其中，每个R c均为羟基。
  5. 根据权利要求1或2所述的含胺转染试剂，其中，Y 1选自C3-C5的亚烷基，每个Y 2均为具有一个羟基取代基的C2-C3的亚烷基。
  6. 根据权利要求1或2所述的含胺转染试剂，其中，每个R a独立地选自C10-C18直链烷基；每个R b独立地为C8-C16直链烷基。
  7. 根据权利要求1所述的含胺转染试剂，其中，该含胺转染试剂选自式(101)-(103)所示结构的化合物及其药学上课接受的盐中的一种或多种：
    Figure PCTCN2019125165-appb-100004
  8. 一种转染复合物，该转染复合物包含关键脂质，所述关键脂质为权利要求1-7中任一项所述的含胺转染试剂。
  9. 根据权利要求8所述的转染复合物，其中，该转染复合物还包含辅助脂质和/或聚乙二醇化脂质。
  10. 根据权利要求9所述的转染复合物，其中，所述关键脂质、辅助脂质和聚乙二醇化脂质之间的摩尔比为(19.7-80):(19.7-80):(0.3-50)；可选地该摩尔比为(50-70):(20-40):(3-30)。
  11. 根据权利要求8-10中任意一项所述的转染复合物，其中，该转染复合物还包含生物活性剂。
  12. 根据权利要求11所述的转染复合物，其中，所述生物活性剂与所述转染复合物中包含的总脂质的重量比为1:1-1:200，所述总脂质指关键脂质、辅助脂质和聚乙二醇化脂质的总和；可选地该重量比为1:1-1:50；可选地该重量比为1:3-1:17。
  13. 根据权利要求11所述的转染复合物，其中，所述生物活性剂选自功能性寡核苷酸或其药学上可接受的盐。
  14. 根据权利要求13所述的转染复合物，其中，所述功能性寡核苷酸为siRNA或其药学上可接受的盐。
  15. 权利要求8-14中任意一项所述的转染复合物在制备用于治疗和/或预防由细胞中特定基因的表达而引起的病理状况或疾病的药物中的用途。
  16. 一种治疗由特定基因的表达而引起的病理状况或疾病的方法，所述方法包含向受试者给予权利要求11-14中任意一项所述的转染复合物。
  17. 一种抑制细胞中特定基因表达的方法，其中，所述方法包含将权利要求11-14中任意一项所述的转染复合物与所述细胞进行接触。
  18. 一种试剂盒，该试剂盒包含权利要求8-14中任意一项所述的转染复合物。
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