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WO2024098361A1 - 人工核酸分子 - Google Patents

人工核酸分子 Download PDF

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Publication number
WO2024098361A1
WO2024098361A1 PCT/CN2022/131289 CN2022131289W WO2024098361A1 WO 2024098361 A1 WO2024098361 A1 WO 2024098361A1 CN 2022131289 W CN2022131289 W CN 2022131289W WO 2024098361 A1 WO2024098361 A1 WO 2024098361A1
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Prior art keywords
nucleic acid
acid sequence
compound
mmol
lipid
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PCT/CN2022/131289
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English (en)
French (fr)
Inventor
张军
王泰云
李航文
董慧芳
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Stemirna Therapeutics Co Ltd
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Stemirna Therapeutics Co Ltd
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Priority to PCT/CN2022/131289 priority Critical patent/WO2024098361A1/zh
Priority to CN202280100916.7A priority patent/CN120092088A/zh
Publication of WO2024098361A1 publication Critical patent/WO2024098361A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of biomedicine.
  • the present invention relates to an artificial nucleic acid molecule, which comprises an open reading frame, a 3'-untranslated region element (3'-UTR element) and/or a 5'-untranslated region element (5'-UTR element).
  • the present invention also relates to a vector comprising a 3'-UTR element and/or a 5'-UTR element, to a cell comprising the artificial nucleic acid molecule or the vector, to a lipid composition or a pharmaceutical composition comprising the artificial nucleic acid molecule or the vector, and to a kit comprising the artificial nucleic acid molecule, the vector, the lipid composition and/or the pharmaceutical composition, preferably for the field of gene therapy and/or gene vaccination.
  • stable RNA For gene therapy and gene vaccination, stable RNA is usually required.Stable RNA can make the product encoded by RNA sequence accumulate in vivo and in its storage process, preparation process and application process, stable RNA can maintain its structural and functional integrity.Therefore, it is necessary to provide stable RNA molecules for gene therapy or gene vaccination to prevent them from experiencing early degradation or decay.And as a scheme for mRNA stabilization, it has been found that naturally occurring eukaryotic mRNA molecules contain specific stabilization elements.For example, its 3'-untranslated region (3'-UTR) and/or 5'-untranslated region (5'-UTR).Both 3'-UTR and 5'-UTR are typical pre-mature (premature) mRNA elements.
  • 3'-UTR 3'-untranslated region
  • 5'-UTR 5'-untranslated region
  • An mRNA molecule carries a gene that encodes a corresponding protein. This gene has specific untranslated regions on both sides at the 5' of the AUG start codon and the 3' of the stop codon. These regions are called 5'-untranslated region and 3'-untranslated region. Usually, 3'-UTR is a part of the sequence between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the polyadenylation sequence, which can regulate the stability, localization and expression of mRNA.
  • ORF open reading frame
  • CDS coding sequence
  • the present invention provides an artificial nucleic acid molecule comprising
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), the 3'-UTR element comprising a nucleic acid sequence selected from the group consisting of:
  • the 3'-UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO:44, and the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO:44; or (ii) wherein the 3'-UTR element comprises a nucleic acid sequence of 3'-UTR derived from transcripts of the following genes or a variant thereof: HCV, CoV2, CVB3, AES and AAT, and the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence derived from; or (iii) wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:16; or the corresponding RNA sequence of the above nucleic acid sequence.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:3.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:4.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:44 and also comprises a nucleic acid sequence of SEQ ID NO:90, 91 or 93.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO: 1, 2 or 5.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the following viral genes or a variant thereof: HCV, CoV2 and CVB3; or from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES or a variant thereof; or from the nucleic acid sequence of the 3'-UTR of the transcript of the human gene AAT or a variant thereof; or from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES or a variant thereof, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element exhibits a length of 3-500 nucleotides, preferably 5-250 nucleotides, more preferably 90-215 nucleotides.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO: 6, 7, 8, 9 or 12.
  • the 3'-UTR element also comprises a nucleic acid sequence of SEQ ID NO:92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:10, 14 or 15.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:12 and also comprises a nucleic acid sequence of SEQ ID NO:9 or 94.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:11 or 13.
  • the artificial nucleic acid molecule further comprises at least one 5'-untranslated region element (5'-UTR element).
  • the 5'-UTR element comprises a nucleic acid sequence of SEQ ID NO:45.
  • the artificial nucleic acid molecule further comprises a 5' cap structure, a polycytidylic acid sequence, a polyadenylic acid sequence or a histone stem-loop.
  • the artificial nucleic acid molecule further comprises a 5' cap structure, a polycytidylic acid sequence, a polyadenylic acid sequence and a histone stem-loop.
  • the ORF is codon optimized.
  • the artificial nucleic acid molecule is RNA, preferably mRNA.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62.
  • the present invention also provides a vector comprising the artificial nucleic acid molecule of the present invention.
  • the present invention also provides a cell comprising the artificial nucleic acid molecule of the present invention or the vector of the present invention.
  • the present invention also provides a lipid composition, which comprises an artificial nucleic acid molecule of the present invention and a lipid that encapsulates the artificial nucleic acid molecule, wherein the lipid that encapsulates the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid; the lipid composition further comprises a cationic polymer, wherein the cationic polymer is associated with the artificial nucleic acid molecule as a complex and is co-encapsulated in the lipid to form a lipid polymer complex.
  • the cationic lipid comprises a lipid compound of formula (I), (II), (III), (IV) or a pharmaceutically acceptable salt thereof, as defined herein.
  • the cationic lipid is M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.
  • the lipid composition comprises 10-70 mol% of a cationic lipid, 10-70 mol% of a phospholipid, 10-70 mol% of a steroid, and 0.05-20 mol% of a polyethylene glycol-modified lipid.
  • the lipid composition comprises 35-50 mol% of a cationic lipid, 10-30 mol% of a phospholipid, 24-44 mol% of a steroid, and 1-1.5 mol% of a polyethylene glycol-modified lipid.
  • the lipid composition comprises 35-50 mol% of cationic lipid, 10-30 mol% of DOPE, 24-44 mol% of cholesterol and 1-1.5 mol% of DMG-PEG.
  • the lipid composition comprises 50 mol% of cationic lipid, 10 mol% of DOPE, 38.5 mol% of cholesterol and 1.5 mol% of DMG-PEG.
  • the lipid composition comprises 40 mol% of cationic lipid, 15 mol% of DOPE, 43.5 mol% of cholesterol and 1.5 mol% of DMG-PEG.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention or the lipid composition of the present invention, and a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention also provides use of the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, the lipid composition of the present invention or the pharmaceutical composition of the present invention in the preparation of a vaccine or a drug for gene therapy.
  • the present invention also provides use of the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, the lipid composition of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing a disease.
  • the present invention also provides a method for increasing the translation efficiency of an artificial nucleic acid molecule, preferably an mRNA molecule or a vector, the method comprising linking an open reading frame to a 3'-UTR element as defined in the present invention.
  • the present invention also provides a kit comprising the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, the lipid composition of the present invention or the pharmaceutical composition of the present invention.
  • the present invention also provides a method for producing an artificial nucleic acid molecule, the method comprising:
  • Figure 1 shows a plasmid map of pUC57-Luc.
  • FIG2 shows exemplary sequences of test artificial nucleic acid molecules.
  • Figure 3 shows the effect of different 3'-UTR elements on the expression of the reporter gene luciferase.
  • the ordinate is the sequence number of the corresponding 3'-UTR elements contained in different artificial nucleic acid molecules, wherein the artificial nucleic acid molecule containing only a poly (A) sequence after the stop codon of the luciferase gene relative to the tested artificial nucleic acid molecule is the reference nucleic acid molecule (ctrl).
  • At least one or “one or more” may mean 1, 2, 3, 4, 5, 6, 7, 8 or more.
  • the expressions “comprises,” “comprising,” “containing,” and “having” are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components.
  • the expression “consisting of” excludes any element, step, or component not specified.
  • the expression “consisting essentially of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions “consisting essentially of” and “consisting of” are encompassed within the meaning of the expression “comprising.”
  • connection term "and/or” between multiple mentioned elements should be understood to include individual and combined options.
  • “and/or” includes “and” and “or”.
  • a and/or B includes A, B and A+B.
  • A, B and/or C include A, B, C and any combination thereof, such as A+B, A+C, B+C and A+B+C. More elements defined by “and/or” are understood in a similar manner and include any one of them and any combination thereof.
  • any numerical value or numerical range such as concentration or concentration range, is understood to be modified by the term "about” in any case. Therefore, numerical values generally include ⁇ 10% of the value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Similarly, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values within the range, including integers and fractions within the range, unless the context clearly indicates otherwise.
  • nucleotide includes deoxyribonucleotides and ribonucleotides and their derivatives.
  • ribonucleotide is a constituent substance of ribonucleic acid (RNA), which is composed of a molecule of base, a molecule of pentose, and a molecule of phosphoric acid. It refers to a nucleotide with a hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group.
  • Deoxyribonucleotide is a constituent substance of deoxyribonucleic acid (DNA), which is also composed of a molecule of base, a molecule of pentose, and a molecule of phosphoric acid. It refers to a nucleotide in which the hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes.
  • Nucleotide is usually referred to by a single letter representing the base: "A (a)” refers to deoxyadenosine or adenylic acid containing adenine, “C (c)” refers to deoxycytidine or cytidine containing cytosine, “G (g)” refers to deoxyguanosine or guanylate containing guanine, “U (u)” refers to uridine containing uracil, and “T (t)” refers to deoxythymidylate containing thymine.
  • polynucleotide and “nucleic acid” are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA).
  • Polynucleotide sequence and “nucleotide sequence” are used interchangeably to refer to the order of nucleotides in a polynucleotide.
  • DNA coding strand sense strand
  • RNA it encodes can be considered to have the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.
  • artificial nucleic acid molecule may be understood as a non-natural nucleic acid molecule. Such nucleic acid molecules may be non-natural due to their individual sequences (which do not exist naturally) and/or due to other modifications (e.g., structural modifications of naturally non-existent nucleotides). Artificial nucleic acid molecules may be DNA molecules, RNA molecules, or hybrid molecules comprising DNA and RNA portions. Typically, artificial nucleic acid molecules may be designed and/or produced by genetic engineering methods. In this case, the artificial nucleic acid molecule comprises an artificial sequence that is normally non-existent naturally, and the artificial sequence is different from the wild-type sequence by at least one nucleotide. The term "wild-type sequence" may be understood as a naturally occurring sequence.
  • RNA can be modified RNA. That is, RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides or linking groups. “Modified” groups can also be referred to as “altered” groups in this article. Groups can be modified or altered chemically, structurally or functionally. For example, a modified nucleobase can include one or more non-naturally occurring substitutions.
  • the term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules into cells, preferably into eukaryotic cells.
  • the term “transfection” includes any method known to those skilled in the art for introducing nucleic acid molecules into cells, preferably into eukaryotic cells, such as into mammalian cells. Such methods include, for example, electroporation, lipofection based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers (e.g., DEAE-dextran or polyethyleneimine), etc.
  • the method is lipofection.
  • expression includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides.
  • translation efficiency refers to a nucleic acid molecule (e.g., mRNA) comprising an open reading frame (ORF). Translation efficiency is experimentally measurable. Translation efficiency is usually measured by measuring the amount of protein translated by the ORF. For experimental measurements of translation efficiency, the ORF preferably encodes a reporter protein or any other quantifiable protein. In the context of the present invention, translation efficiency is particularly useful for such nucleic acid molecules, wherein, in addition to the ORF, at least one 3'-UTR element is also included, preferably as defined herein. It should be understood that in the present invention, high translation efficiency is usually provided by a specific UTR element (specific 3'-UTR element).
  • the ORF appropriately encodes a reporter protein or any other quantifiable protein
  • the present invention is not limited to such purposes; therefore, at least one 3'-UTR element of the present invention (which provides high translation efficiency) can be included in a nucleic acid molecule containing an ORF that does not encode a reporter protein.
  • Translation efficiency is a relative term, which is obtained by determining the translation efficiency of multiple (e.g., two or more) nucleic acid molecules and comparing them, for example, by experiments to quantify the protein encoded by the ORF.
  • One of the nucleic acid molecules can be referred to as a "reference nucleic acid molecule” or “reference construct”, and the other can be referred to as a “test nucleic acid molecule” or “test construct”, and the test nucleic acid molecule can be an artificial nucleic acid molecule as described in the present invention.
  • the reference nucleic acid molecule and the test nucleic acid molecule share the same ORF (same nucleic acid sequence); and preferably, the nucleic acid sequence of the test nucleic acid molecule is the same as that of the reference nucleic acid molecule, except that the UTR element tested, i.e., the 3'-UTR element; in other words, preferably, the test nucleic acid molecule and the reference nucleic acid molecule differ from each other only in that the 3'-UTR element has a different nucleic acid sequence; making the 3'-UTR element the only structural feature that distinguishes the test nucleic acid molecule from the reference nucleic acid molecule.
  • the UTR element tested i.e., the 3'-UTR element
  • vector is a medium for introducing exogenous polynucleotides into host cells, and when the vector is transformed into an appropriate host cell, the exogenous polynucleotides are amplified or expressed.
  • the vector usually remains free, but can be designed to integrate a gene or part thereof into a chromosome of the genome.
  • the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc.
  • Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, poxvirus vectors, and baculovirus vectors, etc.
  • a "cell” is a cell that is used to receive, maintain, replicate and amplify a vector.
  • the cell can also be used to express a polypeptide encoded by the vector.
  • the cell can be a eukaryotic cell or a prokaryotic cell. Suitable cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • an "aliphatic” group is a non-aromatic group in which the carbon atoms are linked in a chain, and may be saturated or unsaturated.
  • alkyl refers to an optionally substituted straight or branched chain saturated hydrocarbon including one or more carbon atoms.
  • C 1 -C 12 alkyl or “C 1-12 alkyl” refers to an optionally substituted straight or branched chain saturated hydrocarbon including 1-12 carbon atoms.
  • alkoxy refers to an alkyl group as described herein, which is connected to the remainder of the molecule through an oxygen atom.
  • alkylene refers to a divalent group formed by the corresponding alkyl group losing one hydrogen atom.
  • alkenyl refers to an optionally substituted straight or branched hydrocarbon comprising two or more carbon atoms and at least one double bond.
  • C 2 -C 12 alkenyl or “C 2-12 alkenyl” refers to an optionally substituted straight or branched hydrocarbon comprising 2 to 12 carbon atoms and at least one carbon-carbon double bond.
  • the alkenyl group may include one, two, three, four or more carbon-carbon double bonds.
  • halogen refers to fluorine, chlorine, bromine and iodine.
  • the term "carbocycle” refers to a monocyclic or polycyclic non-aromatic system comprising one or more rings consisting of carbon atoms.
  • C 3-8 carbocycle means a carbocycle comprising 3-8 carbon atoms.
  • the carbocycle may include one or more carbon-carbon double bonds or triple bonds. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, etc.
  • when the carbocycle is saturated (i.e., without unsaturated bonds), it may also refer to the corresponding cycloalkyl.
  • the carbocycle described herein refers to unsubstituted and substituted, i.e., optionally substituted carbocycles.
  • heterocycle refers to a monocyclic or polycyclic system including one or more rings and including at least one heteroatom.
  • the heteroatom can be, for example, nitrogen, oxygen, phosphorus or sulfur atoms.
  • the heterocycle can include one or more double bonds or triple bonds and can be non-aromatic. Examples of heterocycles include, but are not limited to, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl and piperidinyl.
  • the heterocycle can contain, for example, 3-10 atoms (non-hydrogen), i.e., 3-10 yuan heterocycles (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 yuan), wherein one or more atoms are heteroatoms (e.g., N, O, S or P).
  • 3-10 atoms non-hydrogen
  • 3-10 yuan heterocycles e.g., 3, 4, 5, 6, 7, 8, 9 or 10 yuan
  • one or more atoms are heteroatoms (e.g., N, O, S or P).
  • heterocycle When the heterocycle is saturated (i.e., without unsaturated bonds), it can also refer to the corresponding heterocycloalkyl.
  • heterocycles described herein refer to two types of unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles.
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated ⁇ electron system.
  • a C 6 -C 10 alkylaryl group may have 6-10 carbon atoms, such as 6, 7, 8, 9, 10 carbon atoms.
  • Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.
  • heteroaryl refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring.
  • the heteroaryl group may have 5-10 ring atoms (5-10 membered heteroaryl), including 5, 6, 7, 8, 9 or 10 members, particularly 5 or 6 membered heteroaryl groups.
  • heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.
  • the term "interrupted by one or more groups” means that the one or more groups exist on the carbon chain, and the rest of the carbon chain is connected to both ends of the one or more groups.
  • any of R 1 -R 7 may be optionally substituted.
  • halogen atoms e.g., chloro, bromo, fluoro, or iodo
  • carboxylic acids
  • the substituent itself can be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein.
  • alkyl can be further substituted with one, two, three, four, five, or six substituents as described herein.
  • the term "compound” is intended to include isotopic compounds of the depicted structure.
  • “Isotopes” refer to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus, such as deuterium isotopes.
  • isotopes of hydrogen include tritium and deuterium.
  • the compounds, salts or complexes of the present invention can be prepared in combination with solvents or water molecules to form solvates and hydrates by conventional methods.
  • optionally substituted means that the subsequently described event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.
  • optionally substituted alkyl means that the alkyl group may or may not be substituted, and that the description includes substituted alkyl radicals and unsubstituted alkyl radicals.
  • contacting a mammalian cell with a lipid composition means that a mammalian cell and a lipid nanoparticle are physically connected.
  • the method of contacting a cell with an external entity in vivo and in vitro is well known in the biological field.
  • contacting a lipid composition with a mammalian cell in a mammal can be carried out by different routes of administration (e.g., intravenous, intramuscular, intradermal and subcutaneous) and can relate to different amounts of lipid compositions.
  • the lipid composition can contact more than one mammalian cell.
  • delivery refers to providing an entity to a target.
  • delivering an artificial nucleic acid molecule to a subject may involve administering a lipid composition comprising the artificial nucleic acid molecule to the subject.
  • lipid component is a component of a composition that includes one or more lipids.
  • the lipid component can include one or more cationic lipids, PEGylated lipids, structural lipids, or helper lipids.
  • phrases "pharmaceutically acceptable” is used herein to refer to compounds, salts, materials, compositions and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are consistent with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts refer to derivatives of the disclosed compounds in which the parent compound is altered by converting an existing acid or base moiety into its salt form (e.g., by reacting a free basic group with a suitable organic acid).
  • pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
  • alkali metal or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like; and non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods.
  • these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent, or in a mixture of the two; non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred.
  • gene therapy is understood to be the treatment of a patient's body or an isolated component of a patient's body, such as an isolated tissue/cell, by a nucleic acid encoding a peptide or protein. It can typically include at least one of the following steps: a) directly administering a nucleic acid (preferably an artificial nucleic acid molecule as defined herein) to a patient by any administration route or in vitro to an isolated cell/tissue of a patient, which results in in vivo/ex vivo or in vitro transfection of the patient's cells; b) transcribing and/or translating the introduced nucleic acid molecule; and optionally c) if the nucleic acid is not directly administered to the patient, the isolated, transfected cells are administered to the patient again.
  • a nucleic acid preferably an artificial nucleic acid molecule as defined herein
  • genetic vaccination can typically be understood as vaccination by administering nucleic acid molecules encoding antigens or immunogens or fragments thereof.
  • Nucleic acid molecules can be administered to the subject's body or to isolated cells of the subject. When certain cells of the body are transfected or when isolated cells are transfected, the antigen or immunogen can be expressed by those cells and then presented to the immune system, causing an adaptive (i.e., antigen-specific) immune response.
  • genetic vaccination typically includes at least one of the following steps: a) administering a nucleic acid (preferably an artificial nucleic acid molecule as defined herein) to a subject (preferably a patient), or to isolated cells of a subject (preferably a patient), which usually results in in vivo or in vitro transfection of the subject's cells; b) transcribing and/or translating the introduced nucleic acid molecules; and optionally c) if the nucleic acid is not directly administered to the patient, the isolated, transfected cells are administered to the subject (preferably the patient).
  • a nucleic acid preferably an artificial nucleic acid molecule as defined herein
  • vaccine refers to a composition comprising an active ingredient (e.g., an artificial nucleic acid molecule of the present invention) that can induce an immune response in a vaccinated subject through vaccination.
  • an active ingredient e.g., an artificial nucleic acid molecule of the present invention
  • the immune response induced by it can provide immune protection and is sufficient to prevent and/or alleviate at least one symptom associated with a pathogen or disease infection.
  • treat refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, or reducing the occurrence of one or more symptoms or features of a particular infection, disease, disorder, or condition.
  • Preventing refers to guarding against underlying disease or preventing worsening of symptoms or development of disease.
  • prophylactically or therapeutically effective amount refers to an amount of an agent (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) sufficient to prevent or inhibit the occurrence of a disease or symptom and/or slow down, alleviate, or delay the development or severity of a disease or symptom.
  • the prophylactically or therapeutically effective amount is affected by factors including, but not limited to, the rate and severity of development of the disease or symptom, the age, sex, weight, and physiological condition of the subject, the duration of treatment, and the specific route of administration.
  • the prophylactically or therapeutically effective amount may be administered in one or more doses.
  • the prophylactically or therapeutically effective amount may be achieved by continuous or intermittent administration.
  • an artificial nucleic acid molecule comprising
  • ORF open reading frame
  • At least one 3'-untranslated region element (3'-UTR element).
  • open reading frame is a sequence of nucleotide triplets that can be translated into a peptide or protein.
  • the open reading frame preferably contains a start codon at the end of its 5'-untranslated region element, i.e., a combination of three consecutive nucleotides (ATG) that usually encode the amino acid methionine, and an immediately adjacent region that usually presents a plurality of 3 nucleotides in length.
  • ATG consecutive nucleotides
  • the open reading frame of the present invention is preferably a nucleotide sequence, consisting of a number of nucleotides that can be divided by three, which starts with a start codon (e.g., ATG) and which preferably ends with a stop codon (e.g., TAA, TGA, or TAG).
  • the open reading frame can be isolated or it can be integrated into a longer nucleic acid sequence, such as a vector or mRNA.
  • the open reading frame can also be referred to as a "protein coding region".
  • UTRs Untranslated regions
  • Polynucleotides comprising UTRs may be administered to cells, tissues, or organisms, and one or more regulatory features may be measured using conventional methods.
  • 3'-UTR refers to a portion of an artificial nucleic acid molecule that is located 3' (i.e., "downstream") of an open reading frame and that is not translated into a protein.
  • a 3'-UTR is a portion of an mRNA between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the polyadenylation sequence of the mRNA.
  • ORF open reading frame
  • CDS coding sequence
  • the 3'-UTR of an mRNA is not translated into an amino acid sequence.
  • 3'-UTR plays an important role in the regulation of biological complexity. It can regulate the localization expression of mRNA, regulate the translation of mRNA, and regulate protein-protein interactions (see, e.g., Mayr C. What Are 3'UTRs Doing? Cold Spring Harb Perspect Biol. 2019 Oct 1; 11(10): a034728).
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO:44, and the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO:44.
  • the 3'-UTR element comprises T87C or T94C compared to SEQ ID NO:44.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:3.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:3 and also comprises the nucleic acid sequence of SEQ ID NO:92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:4.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:44 and further comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:6, 7, 9, 12, 90, 91, 92, 93, 95 and 96.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:90.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:1.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:91.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:2.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:93.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:5.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:17.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:95.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:18.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:7.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:25.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44, and also comprises the nucleic acid sequences of SEQ ID NO:96 and 92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:26.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44, and also comprises the nucleic acid sequences of SEQ ID NO:9 and SEQ ID NO:92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:27 or 38.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44, and also comprises the nucleic acid sequences of SEQ ID NO:12 and 92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:29 or 40.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:9.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:37.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:12.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:39.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:44 and also comprises the nucleic acid sequence of SEQ ID NO:6.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:41.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:44 and also comprises a nucleic acid sequence of SEQ ID NO:90, 91 or 93.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO: 1, 2 or 5.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), wherein the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 17, 18, 25, 26, 27, 29, 37, 38, 39, 40 or 41.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO: 1, 2, 3, 4 or 5.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-untranslated region element comprising a nucleic acid sequence derived from the 3'-UTR of a transcript of the following genes or a variant thereof: HCV, CoV2, DENV2, TCV, CYBA, BYDA, CVB3, AES and AAT, the variant comprising truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • nucleic acid sequence derived from the 3'-UTR of the transcript of a gene refers to a nucleic acid sequence based on the 3'-UTR sequence of the transcript of a gene or a fragment or part thereof (preferably a naturally occurring gene or a fragment or part thereof).
  • Nucleic acid sequence derived from the 3'-UTR of the transcript of a gene includes a sequence corresponding to the entire 3'-UTR sequence, i.e., the full-length 3'-UTR sequence of the transcript of the gene, and a sequence corresponding to a fragment of the 3'-UTR sequence of the transcript of the gene.
  • the fragment of the 3'-UTR of the transcript of the gene contains a continuous nucleotide corresponding to a continuous nucleotide in the full-length 3'-UTR of the transcript of the gene, which represents at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%
  • the fragment retains the regulatory function of the translation of the ORF connected to the 3'-UTR or its fragment.
  • truncated refers to a fragment or portion of a nucleic acid sequence based on a 3'-UTR sequence of a transcript of a gene.
  • the truncated 3'-UTR sequence comprises a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length 3'-UTR of the transcript of the gene, which represents at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the full-length 3'-UTR of the transcript of the gene.
  • variant refers to a variant of a 3'-UTR of a naturally occurring gene transcript, preferably a variant of a 3'-UTR of a viral gene transcript, and more preferably a variant of a 3'-UTR of a mammalian gene transcript.
  • the variant may be a modified 3'-UTR of a gene transcript.
  • the variant of the 3'-UTR may present truncation, terminal extension, or one or more nucleotide deletions, additions and/or mutations.
  • the variant of the 3'-UTR of the gene transcript is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identical to the naturally occurring 3'-UTR from which the variant is derived.
  • terminally extended refers to a variant of a 3'-UTR having one or more nucleotides added to its N-terminus or C-terminus compared to the naturally occurring or modified 3'-UTR from which the variant is derived.
  • the 3'-UTR element exhibits a length of at least about 3 nucleotides, preferably at least about 5 nucleotides, more preferably at least about 10, 15, 20, 25 or 30 nucleotides, even more preferably at least about 50 nucleotides, and most preferably at least about 90 nucleotides. In a preferred embodiment, the 3'-UTR element exhibits a length of 3 to about 500 nucleotides, preferably 5 to about 250 nucleotides, and more preferably 90 to 215 nucleotides.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcripts of the following viral genes: HCV, CoV2, DENV2, TCV, CYBA, BYDA and CVB3, and is truncated.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:8, 19, 21 or 23.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES and is truncated.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:9.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the human gene AAT or AES and is truncated.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:12 or 96.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES and is truncated.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:98.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the following viral genes or a variant thereof: HCV, CoV2, DENV2, TCV, CYBA, BYDA and CVB3, wherein the variant comprises truncation and 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:6, 7, 20, 22, 30, 31 or 32.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES or a variant thereof, wherein the variant comprises a truncation and 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:94.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES or a variant thereof, wherein the variant comprises truncation and 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:97.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES or a variant thereof, wherein the variant comprises truncations and terminal extensions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:9 and also comprises the nucleic acid sequence of SEQ ID NO:92 or 12.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:10, 13 or 28.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:9, and also comprises the nucleic acid sequences of SEQ ID NO:92 and 12.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:33.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the human gene AAT or a variant thereof, wherein the variant comprises truncations and terminal extensions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:12, and also comprises the nucleic acid sequence of SEQ ID NO:6, 9, 92, 94, 97 or 98.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:11, 13, 14, 28, 34, 35 or 36.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:12, and also comprises the nucleic acid sequences of SEQ ID NO:9 and 92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:33.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES or a variant thereof, wherein the variant comprises truncations and terminal extensions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:98 and also comprises the nucleic acid sequence of SEQ ID NO:12.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:35.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES or a variant thereof, wherein the variant comprises truncation, terminal extension and 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:94 and also comprises the nucleic acid sequence of SEQ ID NO:12.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:11.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the following viral genes or a variant thereof: HCV, CoV2, DENV2, TCV, CYBA, BYDA and CVB3, wherein the variant comprises truncation, terminal extension and 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:6 and also comprises the nucleic acid sequence of SEQ ID NO:92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:15.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:7 and also comprises the nucleic acid sequence of SEQ ID NO:92.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:24.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:6 and also comprises the nucleic acid sequence of SEQ ID NO:12.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:36.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES or a variant thereof, wherein the variant comprises truncation, terminal extension and 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO:97 and also comprises the nucleic acid sequence of SEQ ID NO:12.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:34.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-untranslated region element comprises a nucleic acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 19, 20, 21, 22, 23, 24, 28, 30, 31, 32, 33, 34, 35, 36, 94, 96, 97 or 98.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-untranslated region element comprising a nucleic acid sequence derived from the 3'-UTR of a transcript of the following genes or a variant thereof: HCV, CoV2, CVB3, AES and AAT, the variant comprising truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the following viral genes or its variants: HCV, CoV2 and CVB3, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES or a variant thereof, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the human gene AAT or a variant thereof, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES or a variant thereof, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the following viral genes or its variant: HCV, CoV2 and CVB3; or derived from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES or its variant; or derived from the nucleic acid sequence of the 3'-UTR of the transcript of the human gene AAT or its variant; or derived from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES or its variant, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), wherein the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO: 16, 42 or 43.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:16.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), the 3'-UTR element comprising a nucleic acid sequence selected from the group consisting of:
  • the 3'-UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO:44, and the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO:44; or
  • the 3'-UTR element comprises a nucleic acid sequence derived from the 3'-UTR of a transcript of the following genes or a variant thereof: HCV, CoV2, DENV2, TCV, CYBA, BYDA, CVB3, AES and AAT, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence derived therefrom; or
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:16, 42 or 43.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), the 3'-UTR element comprising a nucleic acid sequence selected from the group consisting of:
  • SEQ ID NO:1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43.
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), the 3'-UTR element comprising a nucleic acid sequence selected from the group consisting of:
  • the 3'-UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO:44, and the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO:44; or
  • the 3'-UTR element comprises a nucleic acid sequence derived from the 3'-UTR of a transcript of the following genes or a variant thereof: HCV, CoV2, CVB3, AES and AAT, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence derived therefrom; or
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), the 3'-UTR element comprising a nucleic acid sequence selected from the group consisting of:
  • the 3'-UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO:44, and the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO:44; or
  • the 3'-UTR element comprises a nucleic acid sequence derived from the 3'-UTR of a transcript of the following genes or a variant thereof: HCV, CoV2, CVB3, AES and AAT, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence derived therefrom; or
  • the artificial nucleic acid molecule comprises
  • ORF open reading frame
  • 3'-UTR element at least one 3'-untranslated region element (3'-UTR element), the 3'-UTR element comprising a nucleic acid sequence selected from the group consisting of:
  • SEQ ID NO:1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
  • 5'-untranslated region element refers to a portion of an artificial nucleic acid molecule that is located at the 5' end (i.e., "upstream") of an open reading frame and is not translated into a protein.
  • the 5'-UTR starts at the transcription start site and terminates at one nucleotide before the start codon of the open reading frame.
  • the 5'-UTR plays a key role in regulating gene expression.
  • Many cis-regulatory elements may be included in the DNA sequence of the 5'-UTR, which may interact with the transcription machinery to regulate the abundance of messenger RNA (mRNA).
  • the transcribed 5'-UTR is composed of a variety of RNA-based regulatory elements, including a 5' cap structure, a secondary structure, an RNA binding protein motif, an upstream open reading frame (uORF), an internal ribosome entry site, a terminal oligopyrimidine (TOP) tract, and a G-quadruplex.
  • RNA-based regulatory elements including a 5' cap structure, a secondary structure, an RNA binding protein motif, an upstream open reading frame (uORF), an internal ribosome entry site, a terminal oligopyrimidine (TOP) tract, and a G-quadruplex.
  • the artificial nucleic acid molecule further comprises at least one 5'-untranslated region element (5'-UTR element).
  • the 5'-untranslated region element comprises the nucleic acid sequence of SEQ ID NO:45.
  • poly(A) sequence or “poly(A) tail” refers to a nucleotide sequence comprising continuous or discontinuous adenylic acid.
  • the poly(A) sequence is generally located at the 3' end of the RNA, such as the 3' end (downstream) of the 3'-UTR. In some embodiments, the poly(A) sequence does not contain nucleotides other than adenylic acid at its 3' end.
  • the poly(A) sequence can be transcribed by a DNA-dependent RNA polymerase according to the coding sequence of the DNA template during the preparation of the IVT-RNA, or can be linked to the free 3' end of the IVT-RNA, such as the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).
  • the artificial nucleic acid molecule further comprises a poly(A) sequence.
  • polycytidylic acid sequence refers to a nucleotide sequence comprising continuous or discontinuous cytidylic acid.
  • the polycytidylic acid sequence is usually located at the 3' end of the RNA, such as the 3' end (downstream) of the 3'-UTR.
  • the polycytidylic acid sequence does not contain nucleotides other than cytidylic acid at its 3' end.
  • the artificial nucleic acid molecule further comprises a polycytidylic acid sequence.
  • the term “5' cap” generally refers to an N7-methylguanosine structure (also known as “m7G cap”, “m7Gppp-”) attached to the 5' end of the mRNA via a 5' to 5' triphosphate bond.
  • the 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or can be attached to the RNA after transcription using a capping enzyme.
  • the artificial nucleic acid molecule further comprises a 5' cap structure.
  • stem loop (whether it is a histone stem loop or not) can be present in single-stranded DNA, or more commonly in RNA.
  • This structure is also called a hairpin or hairpin loop, and is generally composed of a stem and (terminal) loop in a continuous sequence, wherein the stem is formed by two adjacent completely or partially reverse complementary sequences separated by a short sequence as a spacer to a certain extent, and the short sequence as a spacer becomes the loop of the stem-loop structure.
  • These two adjacent completely or partially reverse complementary sequences can be defined as, for example, stem-loop element stem 1 and stem 2.
  • stem-loop element stem 1 and stem 2 form base pairing with each other, a double-stranded nucleic acid sequence fragment is formed, which contains an unpaired loop formed by a short sequence between the stem-loop element stem 1 and stem 2 on the continuous sequence at its end, thereby forming a stem-loop.
  • histone stem-loop is derived from a histone gene (e.g., from a gene of histone family H1, H2A, H2B, H3, H4), and comprises intramolecular base pairing of two adjacent completely or partially reverse complementary sequences, thereby forming a stem-loop.
  • the artificial nucleic acid molecule further comprises a histone stem-loop.
  • the artificial nucleic acid molecule further comprises a 5' cap structure, a polycytidylic acid sequence, a polyadenylic acid sequence or a histone stem-loop.
  • the artificial nucleic acid molecule further comprises a 5' cap structure and a poly(C) sequence.
  • the artificial nucleic acid molecule further comprises a 5' cap structure and a poly(A) sequence.
  • the artificial nucleic acid molecule further comprises a 5' cap structure, a poly(C) sequence and a histone stem-loop.
  • the artificial nucleic acid molecule further comprises a 5' cap structure, a poly(A) sequence and a histone stem-loop.
  • the artificial nucleic acid molecule further comprises a 5' cap structure, a polycytidylic acid sequence, a polyadenylic acid sequence and a histone stem-loop.
  • the artificial nucleic acid molecule comprises a poly(A) sequence
  • the poly(A) sequence comprises the nucleic acid sequence of SEQ ID NO:46.
  • the artificial nucleic acid molecules defined herein can be prepared using any method known in the art, including synthetic methods, e.g., solid phase synthesis, and in vitro methods, e.g., in vitro transcription reactions or in vivo reactions, e.g., in vivo propagation of DNA plasmids in bacteria.
  • the artificial nucleic acid molecules of the invention can be codon-optimized for the host cells used for expression.
  • the open reading frame (ORF) is codon-optimized.
  • the artificial nucleic acid molecule is RNA.
  • the artificial nucleic acid molecule is mRNA.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89.
  • the 3'-UTR element comprises a nucleic acid sequence of SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62.
  • the mRNA comprises modified nucleotides, wherein the modified nucleotides are selected from one or more of the following nucleotides: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-ur ...
  • the modified nucleotides are selected from one or more of the following nucleotides: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadeno
  • the present invention also provides an expression vector comprising the artificial nucleic acid molecule of the present invention.
  • the expression vector may further comprise additional nucleic acid sequences, such as regulatory sequences and antibiotic resistance genes.
  • the artificial nucleic acid molecule of the present invention may be present in one or more expression vectors.
  • the vector is a DNA vector.
  • the vector is a plasmid vector or a viral vector.
  • the vector is a plasmid vector, such as a pUC57 plasmid vector.
  • the carrier is a circular molecule.
  • the artificial nucleic acid molecule of the present invention is prepared as a recombinant nucleic acid.
  • Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination technology (such as polymerase chain reaction (PCR) technology), etc.
  • the present invention also provides a method for producing an artificial nucleic acid molecule of the present invention, the method comprising:
  • the present invention also provides a method for increasing the translation efficiency of an artificial nucleic acid molecule, preferably an mRNA molecule or a vector, the method comprising linking an open reading frame to a 3'-UTR element as defined in the present invention.
  • the present invention also provides a cell comprising the artificial nucleic acid molecule or vector of the present invention.
  • the artificial nucleic acid molecule or vector of the present invention can be introduced into a suitable cell by various methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.
  • cells are used to express the peptide or protein encoded by the ORF in the artificial nucleic acid molecule of the present invention.
  • examples of cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells).
  • Suitable mammalian host cells include, but are not limited to, human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, such as HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells.
  • the cell is a mammalian cell.
  • the cells are cells isolated from a human subject.
  • the present invention further provides a lipid composition.
  • the lipid composition is a lipid delivery carrier, and the lipid can encapsulate the artificial nucleic acid molecule of the present invention to form nanoparticles, thereby delivering it into an organism.
  • lipid refers to an organic compound comprising a hydrophobic portion and optionally also a hydrophilic portion. Lipids are generally insoluble in water but soluble in many organic solvents. Typically, amphipathic lipids comprising a hydrophobic portion and a hydrophilic portion can be organized into a lipid bilayer structure in an aqueous environment, for example, in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, steroids, and cholesterol esters, etc.
  • lipid nanoparticle refers to a lipid vesicle with a uniform lipid core, which is a particle formed by lipids, and the lipid components undergo intermolecular interactions to form a nanostructured entity.
  • Nucleic acids eg, mRNA
  • lipids are encapsulated in lipids.
  • Particularly preferred lipid compositions can be, for example, lipid multiplexes (LPPs) as described herein. Methods for preparing such compositions can be as described herein.
  • LPPs are particles with a core-shell structure, wherein nucleic acids are contained in multiplexes, and the multiplexes themselves are encapsulated in biocompatible lipid bilayer shells to form lipid nanoparticles of the present invention.
  • the lipid composition of the present invention is a lipid multiplex (LPP).
  • the lipid composition of the present invention is a lipid multiplex (LPP) comprising an artificial nucleic acid molecule.
  • the lipid encapsulating the artificial nucleic acid molecule of the present invention is selected from one or more of the following lipids: cationic lipids, phospholipids, steroids and/or polyethylene glycol-modified lipids.
  • the cationic lipid is an ionizable cationic lipid.
  • the lipid composition of the present invention comprises the artificial nucleic acid molecule of the present invention and a lipid encapsulating the artificial nucleic acid molecule.
  • the lipid encapsulating the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid.
  • the lipid composition comprises a cationic lipid, wherein the cationic lipid comprises DOTMA, DOTAP, DDAB, DOSPA, DODAC, DODAP, DC-Chol, DMRIE, DMOBA, DLinDMA, DLenDMA, CLinDMA, DMORIE, DLDMA, DMDMA, DOGS, N4-cholesteryl-spermine, DLin-KC2-DMA, DLin-MC3-DMA, a compound of formula (I), (II), (III) or (IV) as described herein, or a combination thereof.
  • the cationic lipid comprises M5, MC3, ALC-0315, SM-102.
  • the cationic lipid comprises SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.
  • the cationic lipid comprises M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.
  • the lipid composition comprises a phospholipid and/or a steroid.
  • the lipid composition comprises a phospholipid as described herein, wherein the phospholipid comprises 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn- Glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocho
  • the lipid composition comprises a steroid as described herein, wherein the steroid comprises cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, ⁇ -tocopherol and derivatives thereof.
  • the lipid composition comprises a phospholipid as described herein and a steroid.
  • the lipid composition comprises DOPE.
  • the lipid composition comprises DSPC.
  • the lipid composition comprises cholesterol.
  • the lipid composition comprises DOPE and cholesterol.
  • the lipid composition comprises DSPC and cholesterol.
  • the lipid composition comprises cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipids DOPE and cholesterol.
  • the lipid composition comprises cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipids DSPC and cholesterol.
  • the lipids encapsulating the artificial nucleic acid molecules of the present invention further comprise polyethylene glycol-modified lipids.
  • the polyethylene glycol-modified lipids comprise DMG-PEG (e.g., DMG-PEG 2000), DOG-PEG, and DSPE-PEG or a combination thereof.
  • the polyethylene glycol-modified lipid is DSPE-PEG.
  • the polyethylene glycol-modified lipid is DMG-PEG (e.g., DMG-PEG 2000).
  • the lipid composition comprises a cationic lipid, DOPE, cholesterol, and DSPE-PEG.
  • the lipid composition comprises a cationic lipid, DSPC, cholesterol, and DSPE-PEG.
  • the lipid composition comprises a cationic lipid, DSPC, cholesterol, and DMG-PEG.
  • the lipid composition comprises a cationic lipid, DOPE, cholesterol and DMG-PEG.
  • the lipid composition comprises the cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, DOPE, cholesterol and DMG-PEG.
  • the lipid composition of the present invention further comprises a cationic polymer, which is associated with the artificial nucleic acid molecule as a complex and is co-encapsulated in the lipid.
  • the cationic polymer comprises poly-L-lysine, protamine, polyethyleneimine (PEI), or a combination thereof. In one embodiment, the cationic polymer is protamine. In one embodiment, the cationic polymer is polyethyleneimine.
  • the amount of lipid in the lipid composition is calculated as a mole percent (mol %), which is determined based on the total moles of lipid in the composition.
  • the amount of cationic lipid in the lipid composition is about 10-about 70 mol%. In some embodiments, the amount of cationic lipid in the lipid composition is about 20-about 60 mol%, about 30-about 50 mol%, about 35-about 50 mol%, about 35-about 45 mol%, about 38-about 45 mol%, about 40-about 45 mol%, about 40-about 50 mol%, or about 45-about 50 mol%.
  • the amount of phospholipids in the lipid composition is about 10-about 70 mol%. In one embodiment, the amount of phospholipids in the lipid composition is about 20-about 60 mol%, about 30-about 50 mol%, about 10-about 30 mol%, about 10-about 20 mol%, or about 10-about 15 mol%.
  • the amount of cholesterol in the lipid composition is about 10-about 70 mol%. In one embodiment, the amount of cholesterol in the lipid composition is about 20-about 60 mol%, about 24-44 mol%, about 30-about 50 mol%, about 35-about 40 mol%, about 35-about 45 mol%, about 40-about 45 mol%, or about 45-about 50 mol%.
  • the amount of polyethylene glycol-modified lipids in the lipid composition is about 0.05-about 20 mol%. In one embodiment, the amount of polyethylene glycol-modified lipids in the lipid composition is about 0.5-about 15 mol%, about 1-about 10 mol%, about 5-about 15 mol%, about 1-about 5 mol%, about 1-about 1.5 mol%, about 1.5-about 3 mol%, or about 2-5 mol%.
  • the lipid composition comprises 10-70 mol % of cationic lipids, 10-70 mol % of phospholipids, 10-70 mol % of steroids and 0.05-20 mol % of polyethylene glycol-modified lipids. In a preferred embodiment, the lipid composition comprises 35-50 mol % of cationic lipids, 10-30 mol % of phospholipids, 24-44 mol % of steroids and 1-1.5 mol % of polyethylene glycol-modified lipids.
  • LPP comprises an artificial nucleic acid molecule of the present invention and a lipid encapsulating the artificial nucleic acid molecule, wherein the lipid encapsulating the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid; the LPP further comprises a cationic polymer, wherein the cationic polymer is associated with the artificial nucleic acid molecule as a complex.
  • the lipid composition of the present invention comprises an artificial nucleic acid molecule of the present invention and a lipid encapsulating the artificial nucleic acid molecule, wherein the lipid encapsulating the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid; the lipid composition further comprises a cationic polymer, wherein the cationic polymer is associated with the artificial nucleic acid molecule as a complex, and is co-encapsulated in the lipid to form a lipid polymer complex.
  • the lipid composition comprises 2.5-20 mol% of a polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid composition.
  • the phospholipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or a combination thereof.
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DSPC distearoylphosphatidylcholine
  • the steroid is cholesterol.
  • the cationic polymer is protamine.
  • the polyethylene glycol-modified lipid is selected from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) or a combination thereof.
  • the cationic lipid is selected from M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.
  • the lipid encapsulation complex comprises 50 mol% of M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10 mol% of DOPE, 38.5 mol% of cholesterol and 1.5 mol% of DMG-PEG.
  • the lipid encapsulation complex comprises 40 mol% of M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 15 mol% of DOPE, 43.5 mol% of cholesterol and 1.5 mol% of DMG-PEG.
  • Cationic lipids are lipids that can have a net positive charge at a given pH. Lipids with a net positive charge can associate with nucleic acids through electrostatic interactions.
  • cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), didecyldimethylammonium bromide (DDAB), 2,3-dioleoyloxy-N-[2(spermaminecarboxamide)ethyl]-N,N-dimethyl-l-propylamine trifluoroacetate (2,3-dioleoyloxy-N-[2(spermaminecarboxamide)ethyl]-N,N-dimethyl-l-propylamine trifluoroacetate (2,3-dioleoyloxy-N-[2(spermaminecarboxamide)ethyl]-N,N-dimethyl-l-propylamine trifluoroacetate (2,3-dioleoyloxy-N-
  • DOSPA dioctadecyldimethyl ammonium chloride
  • DODAC 1,2-dioleoyl-3-dimethylammonium-propane
  • DODAP 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol, DC-Chol), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), enyloxy-N,N-dimethyla
  • the cationic lipid is preferably an ionizable cationic lipid.
  • An ionizable cationic lipid has a net positive charge at, for example, acidic pH, but is neutral at higher pH (eg, physiological pH).
  • ionizable cationic lipids include, but are not limited to, dioctadecylamidoglycyl spermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), compounds of formula (I), (II), (III) or (IV) as described herein, or combinations thereof.
  • DOGS dioctadecylamidoglycyl spermine
  • N4-cholesteryl-spermine 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
  • DLin-KC2-DMA 2,2-dilino
  • the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:
  • R1 and R2 are each independently selected from a bond, a C1 - C12 alkyl group, and a C2 - C12 alkenyl group;
  • R3 and R4 are each independently selected from C1 - C12 alkyl, C2 - C12 alkenyl, C6 - C10 aryl and 5-10 membered heteroaryl; and R3 and R4 are each independently optionally substituted by t R6 , t being an integer selected from 1-5;
  • R 6 are each independently selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl
  • M1 and M2 are each independently selected from a bond, H, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, -C(S)S-, a 3-10 membered heterocycle, -NR7- , or
  • R 5 one of M 1 and M 2 , together with the nitrogen atom to which they are attached, form a 3-10 membered heterocyclic ring, and the corresponding R 1 /R 3 or
  • R 2 /R 4 is absent, the heterocyclic ring is optionally substituted by R 7 ;
  • R 5 is selected from C 3-8 carbocycle, -C 1-12 alkylene-Q
  • n are each independently an integer selected from 0-12;
  • the alkyl, alkenyl and alkylene groups are each optionally and independently interrupted by one or more groups selected from: -O-, -S-, -NR 7 -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocycle, and the alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R 7 ;
  • R1 and R2 are each independently selected from C1 - C12 alkyl and C2 - C12 alkenyl, such as C1 - C12 alkyl.
  • one of R1 and R2 is a bond and the other is independently selected from C1 - C12 alkyl and C2 - C12 alkenyl, such as C1 - C12 alkyl.
  • R3 and R4 are each independently selected from C1 - C12 alkyl, C2 - C12 alkenyl, C6 - C10 aryl and 5-10 membered heteroaryl. In another embodiment, R3 and R4 are each independently selected from C1 - C12 alkyl and C2 - C12 alkenyl.
  • R 3 and R 4 may each independently be optionally substituted by t R 6 , t being 1, 2, 3, 4, or 5.
  • R 6 is each independently selected from C 1 -C 12 alkyl.
  • At least one of R 3 and R 4 is C 6 -C 10 aryl or 5-10 membered heteroaryl, for example C 6 -C 10 aryl.
  • R 5 is selected from C 3-8 carbocycle, -C 1-12 alkylene-Q.
  • Q may be selected from H, -OR 7 , -SR 7 , -OC(O)R 7 , -C(O)OR 7 , -N(R 7 )C(O)R 7 , -N(R 7 )S(O) 2 R 7 , -N(R 7 )C(S)R 7 , -N(R 7 ) 2 , cyano, C 3-8 carbocycle, 3-10 membered heterocycle, C 6 -C 10 aryl.
  • the above groups may each be optionally substituted with one or more C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 1 -C 12 alkoxy, C 6 -C 10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo ( ⁇ O), where appropriate.
  • R 5 is selected from C 3-8 carbocycle, -C 1-12 alkylene-Q
  • Q is selected from H, -OR 7 , -SR 7 , -OC(O)R 7 , -C(O)OR 7 , -N(R 7 )C(O)R 7 , -N(R 7 )S(O) 2 R 7 , -N(R 7 )C(S)R 7 , -N(R 7 ) 2 , cyano, C 3-8 carbocycle, 3-10 membered heterocycle, C 6 -C 10 aryl.
  • the above groups may each be optionally substituted with one or more C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 1 -C 12 alkoxy, C 6 -C 10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo ( ⁇ O), where appropriate.
  • R7 can be each independently selected from H, C1 - C12 alkyl, C2- C12 alkenyl, C1 - C12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6 - C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C3-8 carbocycle, preferably selected from H, C1 - C12 alkyl, C2- C12 alkenyl, C1 - C12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6 - C10 aryl and 5-10 membered heteroaryl.
  • each group described above such as C 3-8 carbocycle, -C 1-12 alkylene-Q, includes -OR 7 , -SR 7 , -OC(O)R 7 , -C(O)OR 7 , -N(R 7 )C(O)R 7 , -N(R 7 )S(O) 2 R 7 , -N(R 7 )C(S)R 7 , -N(R 7 ) 2 , C 3-8 carbocycle, 3-10 membered heterocycle, C 6 -C 10 aryl, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 1 -C 12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C 6 -C 10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C The 3-8
  • the alkyl, alkenyl and alkylene groups (e.g., those mentioned in R 1 -R 7 ) in the compounds of formula (I) may each be optionally and independently interrupted by one or more groups selected from: -O-, -S-, -NR 7 -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocycle, and the alkyl, alkenyl and alkylene groups may each be optionally substituted by one or more R 7.
  • the chains (straight or branched) of the alkyl, alkenyl and alkylene groups may each optionally contain one or more groups selected from: -O-, -S-, -NR 7 -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocycle.
  • R7 is each independently selected from H, C1 - C12 alkyl, C2- C12 alkenyl, C1 - C12 alkoxy, carboxylic acid , sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6 - C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C3-8 carbocycle; preferably, R7 is independently selected from H, C1 - C12 alkyl, C2 - C12 alkenyl, C1 - C12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6 - C10 aryl and 5-10 membered heteroaryl.
  • n and n can each independently be an integer selected from 0 to 12, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. When 0 is taken, it means that the corresponding group does not exist.
  • M 1 or M 2 is a bond, the corresponding m or n is not 0, and the carbon chain before M 1 or M 2 is connected to the corresponding R 1 or R 2 .
  • m or n is 0, the corresponding M1 or M2 is not a bond, and the N atom is directly connected to M1 or M2 .
  • M1 or M2 is a bond, the corresponding m or n is 0, and the N atom is directly connected to the corresponding R1 or R2 .
  • M1 and M2 are each independently selected from -C(O)-, -OC(O)-, -C(O)O- and -OC(O)O-. In another embodiment, M1 and M2 are each independently selected from -NR7- , and R7 is as described above.
  • R 5 and one of M 1 and M 2 together with the attached N atom form a 3-10 membered heterocyclic ring, and the corresponding R 1 /R 3 or R 2 /R 4 are absent, and the heterocyclic ring is optionally substituted with R 7 , and R 7 is as described above.
  • R 5 is selected from -C 1-12 alkylene-Q
  • Q is selected from H, -OR 7 , -OC(O)R 7 , -C(O)OR 7 , -N(R 7 )C(O)R 7 , -N(R 7 ) 2 , cyano, and R 7 is as described above.
  • R 1 and R 2 are each independently selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl;
  • R 3 and R 4 are each independently selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl; and R 3 and R 4 are each independently optionally substituted by t R 6 , t being an integer selected from 1-5; and R 6 are each independently selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl.
  • M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)- and -C(S)S-;
  • R 5 is selected from -C 1 -12 alkylene-Q
  • Q is selected from -OR 7 and -SR 7
  • R 7 is independently selected from H, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 1 -C 12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C 6 -C 10 aryl and 5-10 membered heteroaryl;
  • n are each independently an integer selected from 1-12.
  • the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:
  • the cationic lipid comprises M5 or SM-102.
  • the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:
  • the cationic lipid comprises MC3.
  • the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:
  • the cationic lipid comprises ALC-0315.
  • the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:
  • R1 and R2 are each independently selected from C1 - C12 alkyl and C2 - C12 alkenyl;
  • R 3 and R 4 are each independently selected from C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 6 -C 10 aryl and 5-10 membered heteroaryl;
  • R 3 and R 4 are C 6 -C 10 aryl or 5-10 membered heteroaryl, and R 3 and R 4 are each independently optionally substituted by t R 6 , t being an integer selected from 1-5; R 6 are each independently selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl;
  • M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)- and -C(S)S-;
  • R 5 is selected from -C 1-12 alkylene-Q
  • Q is selected from -OR 7 and -SR 7
  • R 7 is independently selected from H, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 1 -C 12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C 6 -C 10 aryl and 5-10 membered heteroaryl;
  • n are each independently an integer selected from 1-12.
  • R 2 is selected from C 1 -C 12 alkyl. In another embodiment, R 2 is selected from C 1 -C 6 alkyl.
  • one of R 3 and R 4 is C 6 -C 10 aryl or 5-10 membered heteroaryl, and the other is C 1 -C 12 alkyl or C 2 -C 12 alkenyl.
  • R3 and R4 are each independently selected from C1 - C12 alkyl and phenyl, provided that at least one of R3 and R4 is phenyl. In another embodiment, one of R3 and R4 is phenyl and the other is C1 - C12 alkyl.
  • R3 and R4 are each independently substituted by t R6 , t being an integer selected from 1-5; for example 1, 2, 3, 4 or 5.
  • t is an integer from 1-3, for example 1, 2 or 3, in particular 1 or 2.
  • each R 6 is independently selected from C 1 -C 12 alkyl, such as C 1 -C 10 alkyl.
  • t is 1 and R 6 is substituted on the phenyl ring at the meta or para position relative to R 1 or R 2 .
  • t is 2 and R 6 is substituted on the phenyl ring at the meta and para positions relative to R 1 or R 2 .
  • R 4 is substituted at the 1st position or the last position of R 2.
  • the 1st position refers to the position of the C atom in R 2 that is directly connected to M 2.
  • the last position refers to the position of the C atom in R 2 that is farthest from M 2.
  • R 4 is selected from C 1 -C 12 alkyl, and R 3 is phenyl.
  • R 3 is substituted at the 1st position or the last position of R 1.
  • the 1st position refers to the position of the C atom in R 1 that is directly connected to M 1.
  • the last position refers to the position of the C atom in R 1 that is farthest from M 1.
  • R 3 is selected from C 1 -C 12 alkyl, and R 4 is phenyl.
  • M1 and M2 are each independently selected from -OC(O)-, -C(O)O- and -OC(O)O-.
  • R 5 is selected from -C 1-5 alkylene-Q, such as C 1 , C 2 , C 3 , C 4 or C 5 alkylene-Q. In an exemplary embodiment, R 5 is selected from -C 1-3 alkylene-Q, such as C 1 , C 2 or C 3 alkylene-Q.
  • Q is selected from -OH and -SH, in particular -OH.
  • m and n are each independently an integer selected from 2-9, such as 2, 3, 4, 5, 6, 7, 8 or 9.
  • m and n are each independently an integer selected from 2-7, such as 2, 3, 4, 5, 6 or 7, more preferably, m and n are each independently an integer selected from 5-7, such as 5, 6 or 7.
  • the compound of formula (I) includes a compound represented by formula (II):
  • R 1 is selected from C 1 -C 6 alkyl
  • R 2 is selected from C 1 -C 10 alkyl
  • R 4 is selected from C 1 -C 10 alkyl
  • M1 and M2 are each independently selected from -OC(O)-, -C(O)O- and -OC(O)O-;
  • R 5 is selected from -C 1-5 alkylene-Q
  • Q is selected from -OR 7 and -SR 7
  • R 7 is independently selected from H, C 1 -C 12 alkyl and C 2 -C 12 alkenyl
  • R 6 are each independently selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl, in particular C 1 -C 12 alkyl;
  • n and n are each independently an integer selected from 2-9, such as 2, 3, 4, 5, 6, 7, 8 or 9;
  • t is an integer selected from 1-3.
  • R 5 is selected from -C 1-3 alkylene-Q
  • Q is selected from -OH and -SH, especially -OH.
  • n and n are each independently an integer selected from 2-7, such as 2, 3, 4, 5, 6 or 7.
  • t is 1 or 2.
  • R 4 is substituted at the 1st position or the last position of R 2.
  • the 1st position refers to the position of the C atom in R 2 that is directly connected to M 2.
  • the last position refers to the position of the C atom in R 2 that is farthest from M 2 .
  • t is 1 and R 6 is substituted on the phenyl ring at the meta or para position relative to R 1 .
  • t is 2 and R 6 is substituted on the phenyl ring at the meta and para positions relative to R 1 .
  • the compound of formula (I) includes a compound represented by formula (III):
  • R 1 is selected from C 1 -C 6 alkyl
  • R 2 is selected from C 1 -C 10 alkyl
  • R 4 is selected from C 1 -C 10 alkyl
  • R 5 is selected from -C 1-3 alkylene-Q, Q is selected from -OH and -SH, especially -OH;
  • t 1 or 2;
  • R 6 is selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl, in particular C 1 -C 12 alkyl;
  • n and n are each independently an integer selected from 2-7, for example 2, 3, 4, 5, 6 or 7.
  • R 4 is substituted at the 1st or last position of R 2.
  • the 1st position refers to the position of R 2 that is The last position refers to the position of the C atom in R 2 that is directly connected to the The position of the C atom that is farthest from the part.
  • t is 1 and R 6 is substituted on the phenyl ring at the meta or para position relative to R 1 .
  • t is 2 and R 6 is substituted on the phenyl ring at the meta and para positions relative to R 1 .
  • the compound of formula (I) includes a compound of formula (IV):
  • R 1 is selected from C 1 -C 6 alkyl
  • R 2 is selected from C 1 -C 10 alkyl
  • R 4 is selected from C 1 -C 10 alkyl
  • t 1 or 2;
  • R 6 are each independently selected from C 1 -C 12 alkyl and C 2 -C 12 alkenyl, in particular C 1 -C 12 alkyl;
  • n and n are each independently an integer selected from 2-7, for example 2, 3, 4, 5, 6 or 7.
  • R 4 is substituted at the 1st or last position of R 2.
  • the 1st position refers to the position of R 2 that is The last position refers to the position of the C atom in R 2 that is directly connected to the The position of the C atom that is farthest from the others.
  • t is 1 and R 6 is substituted on the phenyl ring at the meta or para position relative to R 1 .
  • t is 2 and R 6 is substituted on the phenyl ring at the meta and para positions relative to R 1 .
  • the substituents (eg, R 1 -R 7 ) in the lipid compounds of the present invention do not include alkenyl groups.
  • the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:
  • the cationic lipid comprises the following lipid compound: SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.
  • the cationic lipid comprises the following lipid compound: M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.
  • the lipid composition of the present invention contains phospholipids, which can assist the cell penetration of the lipid composition.
  • phospholipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC).
  • DLPC 1,2-dilinoleoyl-sn-glycero-3-phosphocholine
  • 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetonic acid-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine ethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3
  • the lipid composition of the present invention comprises a steroid, which can serve as a structural component of the lipid composition.
  • steroids examples include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and derivatives thereof.
  • polyethylene glycol-modified lipid or "PEG-modified lipid” or “PEG lipid” refers to a molecule comprising a polyethylene glycol portion and a lipid portion, which is a lipid modified with polyethylene glycol.
  • the PEG lipid can be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG-modified dialkylglycerol, or a combination thereof.
  • examples of polyethylene glycol-modified lipids include, but are not limited to, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-Dioleoyl-rac-glycerol, methoxypolyethylene Glycol (DOGPEG), and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG).
  • DMG-PEG 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol
  • DOGPEG methoxypolyethylene Glycol
  • DSPE-PEG 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol)
  • the polyethylene glycol-modified lipid is DMG-PEG, such as DMG-PEG 2000.
  • DMG-PEG 2000 has the following structure:
  • n 44.
  • cationic polymer refers to any ionic polymer that can carry a net positive charge at a specified pH, thereby electrostatically binding to nucleic acids.
  • examples of cationic polymers include, but are not limited to, poly-L-lysine, protamine, polyethyleneimine (PEI), or a combination thereof.
  • the polyethyleneimine can be linear or branched polyethyleneimine.
  • protamine refers to a low molecular weight basic protein rich in arginine, which exists in sperm cells of various animals (especially fish) and binds to DNA instead of histones.
  • the cationic polymer is protamine (eg, protamine sulfate).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention or the lipid composition of the present invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, softeners, solvents (e.g., water, alcohol, acetic acid and syrup), buffers (e.g., phosphate buffers, histidine buffers and acetate
  • compositions provided herein can be in a variety of dosage forms, including but not limited to solid, semisolid, liquid, powder or lyophilized forms.
  • dosage forms comprising artificial nucleic acid molecules, vectors, cells or lipid compositions
  • preferred dosage forms can generally be, for example, injection solutions and lyophilized powders.
  • compositions provided herein can be administered to a subject by any method known in the art, such as by systemic or topical administration.
  • the route of administration includes, but is not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous or intracavitary), topical (e.g., intratumoral), epidural or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual or topical).
  • parenteral e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous or intracavitary
  • topical e.g., intratumoral
  • epidural or mucosal e.g., intranasal, oral, vaginal, rectal, sublingual or topical
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the present invention relates to use of the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, the lipid composition of the present invention or the pharmaceutical composition of the present invention in the preparation of a vaccine or a medicament for gene therapy.
  • the present invention relates to use of the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, the lipid composition of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing a disease.
  • the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, the lipid composition of the present invention or the pharmaceutical composition of the present invention can be used to treat a disease, a disorder or a condition.
  • the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, the lipid composition of the present invention or the pharmaceutical composition of the present invention can be used to treat a disease, a disorder or a condition characterized by loss or abnormal protein or polypeptide activity.
  • an artificial nucleic acid molecule, a vector, a cell, a lipid composition or a pharmaceutical composition comprising an mRNA encoding a loss or abnormal polypeptide can be administered or delivered to a cell.
  • the mRNA is subsequently translated to produce the polypeptide, thereby reducing or eliminating the problems caused by the absence or abnormal activity of the polypeptide. Since translation can occur rapidly, these methods and artificial nucleic acid molecules, vectors, cells, lipid compositions or pharmaceutical compositions are used to treat acute diseases, disorders or conditions such as sepsis, apoplexy and myocardial infarction.
  • Diseases, disorders or conditions characterized by malfunction or abnormal protein or polypeptide activity to which the artificial nucleic acid molecules, vectors, cells, lipid compositions or pharmaceutical compositions of the present invention can be administered include, but are not limited to, rare diseases, infectious diseases (in the form of vaccines and therapeutic agents), cancers and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.
  • rare diseases e.g., cystic fibrosis
  • genetic diseases e.g., cystic fibrosis
  • autoimmune diseases e.g., cystic fibrosis
  • malfunctioning proteins are missense mutation variants of the cystic fibrosis transmembrane conductance regulatory protein (CFTR) gene, which produce malfunctioning protein variants of the CFTR protein, thereby causing cystic fibrosis.
  • the present invention provides a method for treating such diseases, disorders or conditions of a subject by administering an artificial nucleic acid molecule, vector, cell, lipid composition or pharmaceutical composition of the present invention, wherein the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes the abnormal protein activity present in the subject's cells.
  • the artificial nucleic acid molecule, vector, cell, lipid composition or pharmaceutical composition of the present invention can be administered to a subject using any reasonable amount and any administration route that is effective for the prevention, treatment, diagnosis of a disease, disorder or condition or for any other purpose.
  • the specific amount administered to a given subject may vary depending on the species, age and general condition of the subject; the purpose of administration; the specific composition; the mode of administration, etc.
  • the artificial nucleic acid molecule, vector, cell, lipid composition or pharmaceutical composition of the present invention can be administered to a subject by any method known to those skilled in the art, such as parenteral, oral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous or intraperitoneal.
  • the present invention also provides a kit comprising an artificial nucleic acid molecule, a vector, a cell, a lipid composition or a pharmaceutical composition of the present invention, and instructions for use.
  • the kit may also comprise a suitable container.
  • the kit also comprises a device for administration.
  • the kit generally includes a label indicating the intended use and/or method of use of the contents of the kit.
  • label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • the artificial nucleic acid molecule, vector, cell, lipid composition or pharmaceutical composition of the present invention can exhibit excellent effects, such as but not limited to: 1) improving the translation efficiency of the contained mRNA; and/or 2) the contained mRNA has high stability.
  • the cationic lipid according to formula (I) is synthesized by Silicomai or prepared by reference, such as CN110520409A, WO2018081480A1 or US11,246,933B1; phospholipid (DOPE) is purchased from CordenPharma; cholesterol is purchased from Sigma-Aldrich; mPEG2000-DMG (i.e., DMG-PEG 2000) is purchased from Avanti Polar Lipids, Inc.; PBS is purchased from Invitrogen; protamine sulfate is purchased from Beijing Silian Pharmaceutical Co., Ltd.; mPEG2000-DSPE is purchased from Lipooid GmbH; DSPC is purchased from Avanti Polar Lipids, Inc.
  • DOPE phospholipid
  • DMG-PEG 2000 is purchased from Avanti Polar Lipids, Inc.
  • PBS is purchased from Invitrogen
  • protamine sulfate is purchased from Beijing Silian Pharmaceutical Co., Ltd.
  • mPEG2000-DSPE is purchased from Lipooi
  • LAH Lithium Aluminum Hydride
  • reaction mixture was diluted with DCM (20 mL) and washed with H 2 O (40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether: ethyl acetate (1: 0-20: 1) to give compound 3 (4.365 g, 28%) as a colorless oil.
  • reaction mixture was diluted with DCM (50 mL) and washed with H 2 O (40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.5 g, 45%) as a colorless oil.
  • a mixed solution containing compound 1 (1.22 g, 5.0 mmol, 1.0 eq.), compound 2 (1.30 mg, 10.0 mmol, 2.0 eq.), Pd(PPh 3 ) 4 (289 mg, 0.25 mmol, 0.05 eq.) and K 2 CO 3 (1.38 g, 10.0 mmol, 2.0 eq.) in toluene (10 ml) and H 2 O (1 ml) was stirred at 110° C. under N 2 protection for 1 hour. TLC (petroleum ether:ethyl acetate 19:1) showed that compound 1 was consumed and a new spot was observed.
  • reaction mixture was diluted with DCM (50 mL) and washed with H 2 O (40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.78 g, 62%) as a colorless oil.
  • reaction mixture was diluted with DCM (20 mL) and washed with H 2 O (40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether: ethyl acetate (1: 0-10: 1) to give compound 3 (1.2 g, 66.9%) as a yellow oil.
  • reaction mixture was quenched with H 2 O (80 mL) and extracted with ethyl acetate (60 mL ⁇ 3), and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether/ethyl acetate (1/0-10/1) to obtain yellow oily compound 3 (800 mg, 78%).
  • reaction mixture was diluted with DCM (20 mL) and washed with H 2 O (40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether/ethyl acetate (1/0-10/1) to obtain yellow oily compound 3 (1.2 g, 66.9%).
  • reaction mixture was extracted with ethyl acetate (20 mL) and washed with water (40 mL ⁇ 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether/ethyl acetate (1/0-20/1) to give a colorless oily compound 3 (4.365 g, 28%).
  • reaction mixture was washed with H 2 O (90 mL) and extracted with EA (110 mL) three times, and the organic phase was washed twice with brine (40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography eluting with PE/EA (1/0-30/1) to give compound 3 (1.98 g, 45.5%) as a yellow oil.
  • the residue was purified by silica gel column chromatography eluting with PE/EA (1/0-7/1) to give compound 11 (210 mg, 50.5%) as a yellow oil.
  • reaction mixture was extracted with ethyl acetate (200 mL) and washed with water (200 mL ⁇ 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the residue was purified by silica gel column chromatography, eluted with petroleum ether/ethyl acetate (1/0-20/1) to give a colorless oily compound 3 (7.391 g, 37%).
  • the reaction was directly dried under reduced pressure, and the residue was purified by silica gel column, eluted with DCM/MeOH (1/0-10:1, v/v) to obtain the target product (100 mg, 51%, SW-II-138-2) as a colorless oil.
  • the plasmid containing different 3'-UTR elements is as follows: different 3'-UTR elements (sequences are shown in SEQ ID NO: 1-43) are cloned into the middle region between the downstream stop codon and the poly (A) sequence of the reporter gene firefly luciferase (Firefly Luciferase) by genetic means of homologous recombination, that is, the 3'-UTR region shown in Figure 1, and the rest are the same.
  • a plasmid containing only the poly (A) sequence after the downstream stop codon of firefly luciferase and not containing the tested 3'-UTR element is used as a reference construct.
  • Figure 2 is an exemplary artificial nucleic acid molecule to be tested (SEQ ID NO: 99, wherein the exemplary 3'-UTR element is SEQ ID NO: 1), and the underlined part is the tested 3'-UTR element. Except for the underlined elements, all elements of the sequence shown in Figure 2 are the same as the reference artificial nucleic acid molecule (SEQ ID NO: 100). Therefore, SEQ ID NO:99 differs from SEQ ID NO:100 only in the different 3'-UTR elements tested before the poly(A) sequence.
  • the internal reference plasmid is: the reporter gene firefly luciferase (Firefly Luciferase) is replaced with the renilla luciferase gene (renilla luciferase, Rluc), and the other sequences of the plasmid are the same.
  • Plasmids containing different 3'-UTR elements and internal control plasmids were fully synthesized by Shanghai Sangon Biotechnology Co., Ltd.
  • a DNA template was obtained by PCR amplification using a pair of primers (upstream universal primer: 5’TTGGACCCTCGTACAGAAGCTAATACG 3’; and downstream specific complementary long primer carrying poly(T)) and a high-fidelity DNA polymerase-based PCR amplification kit (Novagen Biotech, Inc.).
  • RNA polymerase was used for co-transcription capping reaction, and RNA was transcribed in vitro to produce Cap1 mRNA.
  • 1-Methyl-pseudouridine-triphosphate was added in the in vitro transcription instead of uridine triphosphate (UTP), so the modification ratio of 1-methyl-pseudouridine in the in vitro transcribed Cap1 mRNA was 100%.
  • DNaseI Thermo Fisher Scientific Inc.
  • Dynabeads Myone was used to purify the mRNA.
  • the purified mRNA was dissolved in 1 mM sodium citrate buffer (pH 6.5+/-0.1), sterile filtered, and frozen at -80°C until use.
  • HEK-293 cells in good growth state were inoculated into 96-well cell culture plates at a density of 3.5 ⁇ 10 4 cells/well, and then placed in a 37°C cell culture incubator for 18-24 hours.
  • Lipofectamine Messenger MAX reagent Thermo Fisher
  • the mRNA of the plasmid containing the reporter gene firefly luciferase and the mRNA of the internal reference control plasmid containing the reporter gene sea cucumber luciferase in Example 3 were transfected into HEK293 cells in a 96-well plate at a mRNA mass ratio of 2:1 per well (a total of 100 ng mRNA), and three replicate wells were set for each sample.
  • the cell plate after mRNA transfection was placed in a 37°C, 5% CO 2 cell culture incubator for 24 hours.
  • the transfected cell samples were tested using the dual luciferase reporter gene detection kit (Novozyme, DL101-01). First, pre-treatment was performed, and the cell lysate was added to the 96-well cell plate at 100 ⁇ L/well. Then, 14 ⁇ L of the lysed cell supernatant was carefully aspirated and transferred to a 96-well all-black ELISA plate, and 70 ⁇ L of firefly luciferase substrate equilibrated to room temperature was added to the ELISA plate, and the plate was quickly mixed and immediately used with an ELISA reader (BioTek).
  • ELISA reader BioTek
  • the relative light unit (RLU) value of firefly luciferase was detected at a wavelength of 560 nm; after the detection was completed, 70 ⁇ L of freshly prepared Renilla luciferase substrate working solution was added to the above reaction solution, and the relative light unit (RLU) value of Renilla luciferase was detected at a wavelength of 480 nm using an ELISA reader (BioTek) immediately after rapid mixing.
  • the actual report for each well is the ratio (Fluc/Rluc) of firefly luciferase RLU (Fluc) to Renilla luciferase RLU (Rluc) in each well.
  • the Fluc/Rluc of the artificial nucleic acid molecule containing only the poly(A) sequence is used as the reference nucleic acid molecule, and the Fluc/Rluc ratio of the artificial nucleic acid molecule containing the different 3'-UTR elements tested relative to the reference is the final relative expression amount.
  • the effects of different 3'-UTR elements on reporter gene expression were analyzed based on the results of three independent repeated experiments, statistical histograms were drawn, and statistical analysis was performed.
  • the experimental results are shown in Figure 3 and Table 2.
  • the translation efficiency of most artificial nucleic acid molecules containing the tested 3'-UTR elements is higher than that of the reference nucleic acid molecules.
  • the relative expression of artificial nucleic acid molecules containing 3'-UTR elements numbered U3006, U3008, U3009, U3010, U3011, U3016, U3020, U3030, U3051, U3053, U3055, U3056, U3057, U3058, U3060 and U3067 is greater than 1.9, and has a higher translation efficiency.

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Abstract

涉及生物医药领域。具体涉及人工核酸分子，其包含至少一个开放阅读框和至少一个3'-非翻译区元件，并且所述人工核酸分子具有高翻译效率。

Description

人工核酸分子 技术领域
本发明属于生物医药领域。本发明涉及人工核酸分子，其包含开放阅读框、3'-非翻译区元件(3'-UTR元件)和/或5'-非翻译区元件(5'-UTR元件)。本发明还涉及包含3'-UTR元件和/或5'-UTR元件的载体，涉及包含所述人工核酸分子或所述载体的细胞，涉及包含所述人工核酸分子或所述载体的脂质组合物或药物组合物，以及涉及包含所述人工核酸分子、所述载体、所述脂质组合物和/或所述药物组合物的试剂盒，优选用于基因治疗和/或基因接种疫苗的领域。
背景技术
对于基因治疗和基因接种疫苗，通常需要稳定的RNA。稳定的RNA能使由RNA序列编码的产物在体内积累并且在其储存过程、制备过程和施用过程中，稳定的RNA能保持其结构和功能完整性。因此，需要提供用于基因治疗或基因接种疫苗的稳定RNA分子，以阻止它们经历早期降解或衰变。而作为mRNA稳定化的方案，已经发现天然存在的真核mRNA分子含有特定的稳定化元件。例如，它的3'-非翻译区(3'-UTR)和/或5'-非翻译区(5'-UTR)。3'-UTR和5'-UTR二者都是典型的成熟前(premature)mRNA元件。
一个mRNA分子携带一个编码相应蛋白的基因。这个基因的两侧在AUG起始密码子的5'处和终止密码子的3'处有特定的不被翻译的区域，这些区域称为5'-非翻译区和3'-非翻译区。通常，3'-UTR是蛋白编码区(开放阅读框(ORF)或编码序列(CDS))和聚腺苷酸序列之间的一部分序列，其可以调控mRNA的稳定性、定位和表达。
现有技术中已经根据一些天然存在的蛋白的非翻译区经过设计和实验研究出许多利于提高mRNA稳定性和表达水平的非翻译区序列。但是本领域仍需要更多更有利于提升基因治疗和基因接种稳定性和安全性的非翻译区序列。
发明内容
在一方面，本发明提供一种人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含选自以下的核酸序列：
(i)其中所述3'-UTR元件包含SEQ ID NO:44所示核酸序列的变体，所述变体与SEQ ID NO:44所示核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者(ii)其中所述3'-UTR元件包含源自以下基因的转录本的3'-UTR的核 酸序列或其变体：HCV、CoV2、CVB3、AES和AAT，所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者(iii)其中所述3'-UTR元件包含SEQ ID NO:16的核酸序列；或者上述核酸序列的相应RNA序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:3的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:4的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:90、91或93的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:1、2或5的核酸序列。
在一实施方案中，所述核酸序列源自以下病毒基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2和CVB3；或源自小鼠基因AES的转录本的3'-UTR的核酸序列或其变体；或源自人基因AAT的转录本的3'-UTR的核酸序列或其变体；或源自牛基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件呈现3-500个核苷酸、优选5-250个核苷酸、更优选90-215个核苷酸的长度。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:6、7、8、9或12的核酸序列。
在一实施方案中，所述3'-UTR元件还包含SEQ ID NO:92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:10、14或15的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:12的核酸序列，并且还包含SEQ ID NO:9或94的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:11或13的核酸序列。
在一实施方案中，所述人工核酸分子还包含至少一个5'-非翻译区元件(5'-UTR元件)。
在一实施方案中，所述5'-UTR元件包含SEQ ID NO:45的核酸序列。
在一实施方案中，所述人工核酸分子还包含5'帽结构、聚胞苷酸序列、聚腺苷酸序列或组蛋白茎环。
在一实施方案中，所述人工核酸分子还包含5'帽结构、聚胞苷酸序列、聚腺苷酸序列和组蛋白茎环。
在一实施方案中，所述ORF是经过密码子优化的。
在一实施方案中，所述人工核酸分子是RNA，优选mRNA。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:47、48、49、50、51、52、53、54、55、56、57、58、59、60、61或62的核酸序列。
在又一方面，本发明还提供一种载体，其包含本发明的人工核酸分子。
在另一方面。本发明还提供一种细胞，其包含本发明的人工核酸分子或本发明的载体。
在另一方面，本发明还提供本一种脂质组合物，其包含本发明的人工核酸分子以及包封所述人工核酸分子的脂质，其中包封所述人工核酸分子的脂质包含阳离子脂质、磷脂、类固醇和聚乙二醇修饰的脂质；所述脂质组合物还包含阳离子聚合物，其中所述阳离子聚合物与所述人工核酸分子缔合为复合物，共同包封在脂质中形成脂质多聚复合物。
在一实施方案中，所述阳离子脂质包含式(I)、(II)、(III)、(IV)的脂质化合物或其药学上可接受的盐，其如本文所限定。在一优选的实施方案中，所述阳离子脂质为M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2。
在一实施方案中，所述脂质组合物包含10-70摩尔％的阳离子脂质、10-70摩尔％的磷脂、10-70摩尔％的类固醇和0.05-20摩尔％的聚乙二醇修饰的脂质。
在一实施方案中，所述脂质组合物包含35-50摩尔％的阳离子脂质、10-30摩尔％的磷脂、24-44摩尔％的类固醇和1-1.5摩尔％的聚乙二醇修饰的脂质。
在一优选的实施方案中，所述脂质组合物包含35-50摩尔％的阳离子脂质、10-30摩尔％的DOPE、24-44摩尔％的胆固醇和1-1.5摩尔％的DMG-PEG。
在一优选的实施方案中，所述脂质组合物包含50摩尔％的阳离子脂质、10摩尔％的DOPE、38.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG。
在一优选的实施方案中，所述脂质组合物包含40摩尔％的阳离子脂质、15摩尔％的DOPE、43.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG。
在又一方面，本发明还提供一种药物组合物，其包含本发明的人工核酸分子，本发明的载体、本发明的细胞或本发明的脂质组合物，以及药学上可接受的载剂、赋形剂或稀释剂。
在又一方面，本发明还提供本发明的人工核酸分子，本发明的载体、本发明的细胞、本发明的脂质组合物或本发明的药物组合物在制备疫苗或用于基因治疗的药物中的用途。
在又一方面，本发明还提供本发明的人工核酸分子，本发明的载体、本发明的细胞、本发明的脂质组合物或本发明的药物组合物在制备用于治疗或预防疾病的药物中的用途。
在又一方面，本发明还提供一种用于增加人工核酸分子、优选mRNA分子或载体的翻译效率的方法，所述方法包括连接开放阅读框与本发明所定义的3'-UTR元件。
在又一方面，本发明还提供一种试剂盒，其包含本发明的人工核酸分子，本发明的载体、本发明的细胞、本发明的脂质组合物或本发明的药物组合物。
在又一方面，本发明还提供一种产生人工核酸分子的方法，所述方法包括：
a)合成本发明的人工核酸分子；或者
b)通过本发明的载体合成人工核酸分子。
附图说明
图1示出pUC57-Luc质粒图谱。
图2示出受试人工核酸分子的示例性序列。
图3示出不同3'-UTR元件对报告基因荧光素酶表达的影响。纵坐标为不同人工核酸分子所包含的相应3'-UTR元件的序列号，其中相对于受试人工核酸分子，在荧光素酶基因终止密码子后仅包含poly(A)序列的人工核酸分子为参比核酸分子(ctrl)。
具体实施方式
定义
本文引用的所有专利、专利申请、科学出版物、制造商的说明书和指南等，无论上文或下文，均整体援引加入本文。本文中的任何内容均不应理解为承认本公开无权先于这样的公开。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文所用，“至少一个(种)”或“一个(种)或多个(种)”可以表示1、2、3、4、5、6、7、8个(种)或更多个(种)。
如本文所用，表述“包括”、“包含”、“含有”和“具有”是开放式的，表示包括所列举的元素、步骤或组分但不排除其他未列举的元素、步骤或组分。表述“由……组成”不包括未指定的任何元素、步骤或组分。表述“基本上由……组成”是指范围限于指定的元素、步骤或组分，加上不显著影响要求保护的主题的基本和新颖性质的任选存在的元素、步骤或组分。应当理解，表述“基本上由……组成”和“由……组成”涵盖在表述“包括”的含义之内。
如本文所用，多个述及的元素之间的连接术语“和/或”应理解为包括单独的和组合的选项。换言之，“和/或”包括“和”以及“或”。例如，A和/或B包括A、B以及A+B。A、B和/或C包括A、B、C及其任意组合，例如A+B、A+C、B+C以及A+B+C。以“和/或”限定的更多元素以类似方式理解，并且包括其中的任一种及其任意组合。
除非另有说明，否则任何数值或数值范围，例如浓度或浓度范围，在任何情况下均应理解为由术语“约”修饰。因此，数值通常包括所述值的±10％。例如，1mg/mL的浓度包括0.9mg/mL至1.1mg/mL。同样的，1％至10％(w/v)的浓度范围包括0.9％(w/v)至11％(w/v)。如本文所用，数值范围的使用明确地包括所有可能的子范围，该范围内的所有单个数值，包括该范围内的整数和分数，除非上下文另外明确指出。
在本文中，“核苷酸”包括脱氧核糖核苷酸和核糖核苷酸及其衍生物。如本文所用，“核糖核苷酸”是核糖核酸(RNA)的构成物质，由一分子碱基，一分子五碳糖，一分子磷 酸组成，其是指在β-D-呋喃核糖(β-D-ribofuranosyl)基团的2’位置具有羟基的核苷酸。而“脱氧核糖核苷酸”是脱氧核糖核酸(DNA)的构成物质，也由一分子碱基，一分子五碳糖，一分子磷酸构成，其是指在β-D-呋喃核糖(β-D-ribofuranosyl)基团的2’位置的羟基被氢取代的核苷酸，是染色体的主要化学成分。“核苷酸”通常由代表其中碱基的单字母来指代：“A(a)”指含有腺嘌呤的脱氧腺苷酸或腺苷酸，“C(c)”指含有胞嘧啶的脱氧胞苷酸或胞苷酸，“G(g)”指含有鸟嘌呤的脱氧鸟苷酸或鸟苷酸，“U(u)”指含有尿嘧啶的尿苷酸，“T(t)”指含有胸腺嘧啶的脱氧胸苷酸。
如本文所用，术语“多核苷酸”和“核酸”可以互换使用，用于指脱氧核糖核苷酸的聚合物(脱氧核糖核酸，DNA)或核糖核苷酸的聚合物(核糖核酸，RNA)。“多核苷酸序列”、“核酸序列”和“核苷酸序列”可以互换使用，用来表示多核苷酸中核苷酸的排序。本领域人员应当理解，DNA编码链(有义链)与其编码的RNA可以看作具有相同的核苷酸序列，DNA编码链序列中的脱氧胸苷酸对应其编码的RNA序列中的尿苷酸。
如本文所用，“人工核酸分子”可以理解为非天然的核酸分子。这种核酸分子可以由于其个体序列(其天然不存在)和/或由于其它修饰(例如天然不存在的核苷酸的结构修饰)而是非天然的。人工核酸分子可以是DNA分子、RNA分子或包含DNA和RNA部分的杂合分子。典型地，可以通过遗传工程方法设计和/或产生人工核酸分子。在这种情况下，人工核酸分子包含通常是天然不存在的人工序列，所述人工序列与野生型序列至少有一个核苷酸不同。术语“野生型序列”可以理解为天然存在的序列。
如本文所用，“修饰过的”指非天然的。例如，RNA可以是修饰过的RNA。也就是说，RNA可以包括一个或多个非天然存在的核碱基、核苷、核苷酸或连接基团。“修饰过的”基团在本文中还可以称为“改变的”基团。基团可以在化学上、结构上或功能上进行修饰或改变。例如，修饰过的核碱基可以包括一个或多个非天然存在的取代。
如本文所用，术语“转染”指将核酸分子，如DNA或RNA(例如mRNA)分子引入细胞，优选引入真核细胞。在本发明范围内，术语“转染”包括本领域技术人员已知的用于将核酸分子引入细胞，优选引入真核细胞，如引入哺乳动物细胞的任何方法。这种方法包括例如，电穿孔、例如基于阳离子脂质和/或脂质体的脂转染、磷酸钙沉淀、基于纳米粒子的转染、基于病毒的转染、或基于阳离子聚合物(如DEAE-葡聚糖或聚乙烯亚胺)的转染等。优选地，所述方法为脂转染。
如本文所用，术语“表达”包括核苷酸序列的转录和/或翻译。因此，表达可以涉及转录物和/或多肽的产生。
如本文所用，术语“翻译效率”涉及包含开放阅读框(ORF)的核酸分子(例如，mRNA)。翻译效率在实验上是可测量的。翻译效率通常通过测定由ORF翻译的蛋白的量而测量。对于翻译效率的实验测量，ORF优选编码报告蛋白或任意其他可以定量的蛋白。在本发明的情形中，翻译效率特别用于这样的核酸分子，其中，除了ORF之外，还包含至少一个3'-UTR元件，优选如本文定义。应该理解，在本发明中，高翻译效率通常由特定 的UTR元件(特定的3'-UTR元件)提供。尽管就实验定量翻译效率来说，ORF适当地编码报告蛋白或任意其他可以定量的蛋白，但是，本发明不限于此类目的；因此，本发明的至少一个3'-UTR元件(其提供高翻译效率)可以被包含在含有不编码报告蛋白的ORF的核酸分子中。
翻译效率是相对术语，其通过确定多种(例如，两种或更多种)核酸分子的翻译效率并进行比较得出，例如，通过实验以定量由ORF编码的蛋白而确定和比较。可以将核酸分子中的一种称为“参比核酸分子”或“参比构建体”，并将另一种称为“受试核酸分子”或“受试构建体”，受试核酸分子可以是本发明所述的人工核酸分子。为了这一目的，参比核酸分子和受试核酸分子共有相同的ORF(相同的核酸序列)；并且优选地，受试核酸分子的核酸序列与参比核酸分子的核酸序列相同，不同之处在于测试的UTR元件，即，3'-UTR元件；换言之，优选地，受试核酸分子和参比核酸分子彼此的差异仅在于3'-UTR元件具有不同的核酸序列；使得3'-UTR元件成为区分所述受试核酸分子和参比核酸分子的唯一的结构特征。
如本文所用，“载体”是用于将外源多核苷酸导入宿主细胞的媒介，当载体转化入适当的宿主细胞时，外源多核苷酸得以扩增或表达。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。如本文所用，载体的定义涵盖质粒、线性化质粒、病毒载体、粘粒、噬菌体载体、噬菌粒、人工染色体(例如，酵母人工染色体和哺乳动物人工染色体)等。病毒载体包括但不限于逆转录病毒载体(包括慢病毒载体)、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体和杆状病毒载体等。
如本文所用，“细胞”是用于接受、保持、复制和扩增载体的细胞。细胞还可以用来表达载体所编码的多肽。当细胞分裂时，载体中所含的核酸复制，从而扩增核酸。细胞可以是真核细胞或原核细胞。合适的细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
如本文所使用，“脂肪族”基团是其中碳原子连接成链的非芳香族基团，并且可以是饱和或不饱和。
如本文所用，术语“烷基”指包括一个或多个碳原子的任选被取代的直链或分支链饱和烃。术语“C 1-C 12烷基”或“C 1-12烷基”指包括1-12个碳原子的任选被取代的直链或分支链饱和烃。如本文所用，术语“烷氧基”指本文所述的烷基，其通过氧原子连接至分子的剩余部分。术语“亚烷基”指失去一个氢原子的相应烷基形成的二价基团。术语“C 1-C 12亚烷基”或“C 1-12亚烷基”指包括1-12个碳原子的任选被取代的直链或分支链亚烷基。
如本文所用，术语“烯基”指包括两个或更多个碳原子和至少一个双键的任选被取代的直链或分支链烃。术语“C 2-C 12烯基”或“C 2-12烯基”指包括2-12个碳原子和至少一个碳-碳双键的任选被取代的直链或分支链烃。烯基可以包括一个、两个、三个、四个或更多个碳-碳双键。
如本文所用，术语“卤素”指氟、氯、溴和碘。
如本文所使用，术语“碳环”指包括一个或多个由碳原子构成的环的单环或多环非芳香系统。术语“C 3-8碳环”意思指包括3-8个碳原子的碳环。碳环可以包括一个或多个碳-碳双键或三键。碳环的实例包括但不限于环丙基、环戊基、环己基等。如本文所使用，当碳环为饱和时(即，不含不饱和键)，也可以指代相应的环烷基。除非另外具体说明，否则本文所述的碳环是指未取代和被取代，即，任选被取代的碳环。
如本文所使用，术语“杂环”指包括一个或多个环且包括至少一个杂原子的单环或多环系统。杂原子可以是例如氮、氧、磷或硫原子。杂环可以包括一个或多个双键或三键并且可以是非芳香族的。杂环的实例包括但不限于咪唑烷基、噁唑烷基、噻唑烷基、吡唑烷基、异噁唑烷基、异噻唑烷基、吗啉基、吡咯烷基、四氢呋喃基和哌啶基。杂环可以包含例如3-10个原子(非氢)，即3-10元杂环(例如3、4、5、6、7、8、9或10元)，其中一个或多个原子为杂原子(例如N、O、S或P)。当杂环为饱和时(即，不含不饱和键)，也可以指代相应的杂环烷基。除非另外具体说明，否则本文所述的杂环是指未取代和被取代的杂环基团两种，即，任选被取代的杂环。
如本文所用，术语“芳基”是指具有共轭的π电子体系的全碳单环或稠合多环的芳香环基团。例如，C 6-C 10烷基芳基可以具有6-10个碳原子，例如6、7、8、9、10个碳原子。芳基的实例包括但不限于苯基、萘基等。
如本文所用，术语“杂芳基”是指单环或稠合多环体系，其中含有至少一个选自N、O、S的环原子，其余环原子为C，并且具有至少一个芳香环。杂芳基可以具有5-10个环原子(5-10元杂芳基)，其包括5、6、7、8、9或10元，特别是5或6元杂芳基。杂芳基的实例包括但不限于吡咯基、呋喃基、噻吩基、咪唑基、噁唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、喹啉基、异喹啉基、四唑基、三唑基、三嗪基、苯并呋喃基、苯并噻吩基、吲哚基、异吲哚基等。
如本文所用，术语“被一个或多个基团中断”是指碳链上存在此一个或多个基团，并且碳链的其余部分与所述一个或多个基团两端相连。
除非另外具体说明，否则本文所述的基团(例如，R 1-R 7中的任一个，如烷基、亚烷基、烯基、芳基、氨基等)可以任选被取代。可选取代基可以选自以下，但不限于：卤素原子(例如氯基、溴基、氟基或碘基)、羧酸(例如-C(O)OH)、醇(例如羟基，-OH)、酯(例如-C(O)OR或-OC(O)R)、醛(例如-C(O)H)、羰基(例如-C(O)R，或由C＝O表示)、酰基卤(例如-C(O)X，其中X是选自溴、氟、氯和碘的卤基)、碳酸酯基(例如-OC(O)OR)、烷氧基(例如-OR)、缩醛(例如-C(OR) 2R””，其中各OR是相同或不同的烷氧基并且R””是烷基或烯基)、磷酸根(例如P(O) 4 3-)、硫醇(例如-SH)、亚砜(例如-S(O)R)、亚磺酸(例如-S(O)OH)、磺酸(例如-S(O) 2OH)、硫醛(例如-C(S)H)、硫酸根(例如S(O) 4 2-)、磺酰基(例如-S(O) 2-)、酰胺(例如-C(O)NR 2或-N(R)C(O)R)、叠氮基(例如-N 3)、硝基(例如-NO 2)、氰基(例如-CN)、异氰基(例如-NC)、酰氧基(例如-OC(O)R)、氨基(例如-NR 2、NRH或-NH 2)、氨甲酰基(例如-OC(O)NR 2、-OC(O)NRH或-OC(O)NH 2)、磺酰胺(例如-S(O) 2NR 2、-S (O) 2NRH、-S(O) 2NH 2、-N(R)S(O) 2R、-N(H)S(O) 2R、-N(R)S(O) 2H、-N(H)S(O) 2H)、C 1-C 12烷基、C 2-C 12烯基、C 6-C 10芳基、5-10元杂芳基或3-10元杂环。在前述任一种中，R各自独立地可以是如本文所定义的取代基，如烷基、烷氧基、亚烷基、卤素、碳环、杂环、芳基、杂芳基、烯基。在一些实施方案中，取代基本身可以进一步被例如一个、两个、三个、四个、五个或六个如本文所定义的取代基取代。例如，烷基可以进一步被一个、两个、三个、四个、五个或六个如本文所述的取代基取代。
如本文所用，术语“化合物”意在包括所描绘结构的同位素化合物。“同位素”是指具有相同原子数但因核中的中子数量不同而具有不同质量数的原子，例如氘同位素。例如，氢的同位素包括氚和氘。另外，本发明的化合物、盐或复合物可以与溶剂或水分子组合制备以通过常规方法形成溶剂化物和水合物。
术语“任选”或“任选地”(例如，任选地被取代)是指随后描述的事件可能会或可能不会发生，并且描述包括所述事件或情况发生的实例以及所述事件或情况不发生的实例。例如，“任选取代的烷基”是指烷基可以被取代，也可以不被取代，并且该描述包括取代的烷基自由基和没有取代的烷基自由基。
应理解，当化学基团按特定顺序书写时，除非另外说明，还涵盖了相反顺序。例如在M 1定义为-C(O)NH-的通式-(R) i-(M1) k-(R) m-(即，-(R) i-C(O)-NH-(R) m-)中，除非另外说明，还涵盖了M 1为-NHC(O)-的化合物(即，-(R) i-NHC(O)-(R) m-)。
如本文所用，术语“接触”指在两个或更多个实体之间建立物理连接。例如，使哺乳动物细胞与脂质组合物接触意味着，使哺乳动物细胞和脂质纳米颗粒共有物理连接。使细胞与外部实体在体内和离体接触的方法是生物领域中众所周知的。例如，使脂质组合物与处于哺乳动物体内的哺乳动物细胞接触可以通过不同施用途径(例如静脉内、肌内、皮内和皮下)进行并且可以涉及不同量的脂质组合物。此外，脂质组合物可以接触超过一个哺乳动物细胞。
如本文所用，术语“递送”指将实体提供至目标。例如，将人工核酸分子递送至受试者可涉及将包括该人工核酸分子的脂质组合物施用给该受试者。
如本文所用，“脂质组分”是包括一种或多种脂质的组合物的组分。例如，脂质组分可以包括一种或多种阳离子脂质、聚乙二醇化脂质、结构脂质或辅助脂质。
短语“药学上可接受的”在本文中用于指在合理的医学判断范围内、适于与人类和动物组织接触使用而无过度毒性、刺激、过敏反应或其它问题或并发症，并且与合理的效益/风险比相符的化合物、盐、材料、组合物和/或剂型。
如本文所用，“药学上可接受的盐”是指所公开化合物的衍生物，其中母体化合物通过将现有酸或碱部分转化成其盐形式(例如通过使游离碱性基团与适合有机酸反应)而改变。药物可接受的盐的实例包括但不限于碱性残基如胺的无机或有机酸盐；酸性残基如羧酸的碱金属或有机盐等。代表性酸加成盐包括但不限于乙酸盐、己二酸盐、褐藻酸盐、抗坏血酸盐、天冬氨酸盐、苯磺酸盐、苯甲酸盐、硫酸氢盐、硼酸盐、丁酸盐、樟脑酸 盐、樟脑磺酸盐、柠檬酸盐、环戊烷丙酸盐、二葡糖酸盐、十二烷基硫酸盐、乙烷磺酸盐、反丁烯二酸盐、葡庚糖酸盐、甘油磷酸盐、半硫酸盐、庚酸盐、己酸盐、氢溴酸盐、盐酸盐、氢碘酸盐、2-羟基-乙烷磺酸盐、乳糖醛酸盐、乳酸盐、月桂酸盐、月桂基硫酸盐、苹果酸盐、顺丁烯二酸盐、丙二酸盐、甲烷磺酸盐、2-萘磺酸盐、烟碱酸盐、硝酸盐、油酸盐、草酸盐、棕榈酸盐、双羟萘酸盐、果胶酸盐、过硫酸盐、3-苯基丙酸盐、磷酸盐、苦味酸盐、特戊酸盐、丙酸盐、硬脂酸盐、琥珀酸盐、硫酸盐、酒石酸盐、硫氰酸盐、甲苯磺酸盐、十一烷酸盐、戊酸盐等。代表性碱金属或碱土金属盐包括但不限于钠、锂、钾、钙、镁盐等；以及无毒铵、季铵和胺阳离子，包括但不限于铵、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、三乙胺、乙胺等。本发明的药学上可接受的盐包括例如由无毒无机或有机酸形成的母体化合物的常规无毒盐。本发明的药学上可接受的盐可以由含有碱性或酸性部分的母体化合物通过常规化学方法合成。一般而言，这些盐可以通过使这些化合物的游离酸或碱形式与化学计算量的量的适当碱或酸在水中或在有机溶剂中，或在这两种的混合物中反应来制备；一般优选非水性介质，如乙醚、乙酸乙酯、乙醇、异丙醇或乙腈。
如本文所用，“基因治疗”可理解为通过编码肽或蛋白的核酸治疗患者的身体或患者身体的分离的组成部分，例如分离的组织/细胞。其典型地可以包括至少一个以下步骤：a)直接将核酸(优选如本文限定的人工核酸分子)通过任何给药途径施用于患者或体外施用于患者的分离的细胞/组织，其导致体内/离体或体外转染患者的细胞；b)转录和/或翻译引入的核酸分子；以及任选地c)如果核酸未直接施用于患者，则将分离、转染的细胞再施用于患者。
如本文所用，“基因疫苗接种”可以典型地理解为通过施用编码抗原或免疫原或其片段的核酸分子进行的疫苗接种。核酸分子可以施用于受试者的身体或施用于受试者的分离的细胞。当转染身体的某些细胞或当转染分离的细胞后，抗原或免疫原可以由那些细胞表达并随后呈递到免疫系统，引起适应性的(即抗原特异性的)免疫应答。因此，基因疫苗接种典型地包括至少一个以下步骤：a)将核酸(优选如本文限定的人工核酸分子)施用给受试者(优选患者)，或施用于受试者(优选患者)的分离的细胞，其通常导致体内或体外转染受试者的细胞；b)转录和/或翻译引入的核酸分子；以及任选地c)如果核酸未直接施用于患者，则将分离、转染的细胞再施用于受试者(优选患者)。
如本文所用，“疫苗”是指包含活性成分(例如本发明的人工核酸分子)的组合物，它能通过接种引起接种对象免疫应答。在具体实施方案中，它诱导的免疫应答能提供免疫保护，并足以预防和/或减轻与病原体或疾病感染相关的至少一种症状
如本文所用，术语“治疗”是指部分或完全地减轻、改良、改善、缓解特定感染、疾病、病症或病况的一种或多种症状或特征，延迟其发作，抑制其进展，降低其严重程度或减少其发生。“预防”指防范潜在疾病或防范症状恶化或疾病发展。
术语“预防或治疗有效量”是指足以预防或抑制疾病或症状的发生和/或减缓、减轻、 延迟疾病或症状的发展或严重程度的试剂(例如核酸、药物、组合物、治疗剂、诊断剂、预防剂等)的量。预防或治疗有效量受到包括但不限于以下因素的影响：疾病或症状的发展速度和严重程度，受试者的年龄、性别、体重和生理状况，治疗的持续时间以及具体施用途径。预防或治疗有效量可以在一个或多个剂量中施用。预防或治疗有效量可以通过持续或间断施用实现。
人工核酸分子
本文提供一种人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)。
如本文所用，“开放阅读框(ORF)”是一些核苷酸三联体的序列，其可以翻译为肽或蛋白。开放阅读框优选在其5'-非翻译区元件末端含有起始密码子，即通常编码氨基酸甲硫氨酸的三个连续的核苷酸的组合(ATG)，和通常呈现多个3核苷酸长度的紧接的区域。本发明的开放阅读框优选为核苷酸序列，由可以被三整除的很多核苷酸组成，其以起始密码子(例如ATG)起始并且其优选以终止密码子(例如，TAA，TGA，或TAG)终止。开放阅读框可以是分离的或可以将其整合入更长的核酸序列，例如载体或mRNA中。还可以将开放阅读框称为“蛋白编码区”
非翻译区(UTR)可具有提供调控作用的特征，例如稳定性、定位和/或翻译效率的增加或降低。可将包含UTR的多核苷酸施用至细胞、组织或生物体，并且可使用常规方法测量一种或多种调控特征。
如本文所用，术语“3'-UTR”是指人工核酸分子的一部分，其位于开放阅读框的3'(即“下游”)，并且其不翻译为蛋白。通常，3'-UTR是mRNA的蛋白编码区(开放阅读框(ORF)或编码序列(CDS))和聚腺苷酸序列之间的一部分mRNA。mRNA的3'-UTR不翻译为氨基酸序列。3'-UTR在生物复杂性的调节中发挥重要作用，其可以调控mRNA的定位表达，可以调控mRNA的翻译，也可以调控蛋白-蛋白相互作用(参见，例如Mayr C.What Are 3'UTRs Doing？Cold Spring Harb Perspect Biol.2019Oct 1；11(10):a034728)。
在一实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含SEQ ID NO:44所示核酸序列的变体，所述变体与SEQ ID NO:44所示核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件与SEQ ID NO:44相比，包含T87C或T94C。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:3的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:3的核酸序列，并且还包含SEQ ID NO:92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:4的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含至少一个选自以下的核酸序列：SEQ ID NO:6、7、9、12、90、91、92、93、95和96。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:90的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:1的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:91的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:2的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:93的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:5的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:17的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:95的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:18的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:7的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:25的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:96和92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:26的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:9和SEQ ID NO:92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:27或38的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:12和92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:29或40的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:9的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:37的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:12的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:39的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:6的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:41的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:90、91或93的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:1、2或5的核酸序列。
在一实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含SEQ ID NO:1、2、3、4、5、17、18、25、26、27、29、37、38、39、40或41的核酸序列。
在一优选实施方案中，所述3'-UTR元件包含SEQ ID NO:1、2、3、4或5的核酸序列。
在一实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含源自以下基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、DENV2、TCV、CYBA、BYDA、CVB3、AES和AAT，所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
术语“源自基因的转录本的3'-UTR的核酸序列”是指基于基因的转录本或其片段或部分(优选天然存在的基因或其片段或部分)的3'-UTR序列的核酸序列。“源自基因的转录本的3'-UTR的核酸序列”包括对应于整个3'-UTR序列的序列，即基因的转录本的全长3'-UTR序列，和对应于基因的转录本的3'-UTR序列的片段的序列。优选地，基因的转录本的3'-UTR的片段包含与基因的转录本的全长3'-UTR中的一段连续核苷酸对应的一段连续核苷酸，其代表基因的转录本的全长3'-UTR的至少5％、至少10％、至少20％、至少30％、至少40％、至少50％|、至少60％、至少70％、至少80％或至少90％。在本文中，优选地，片段保留对与3'-UTR或其片段连接的ORF的翻译的调节功能。
术语“截短的”是指所基于基因的转录本的3'-UTR序列的核酸序列的片段或部分。优选地，截短的3'-UTR序列包含与基因的转录本的全长3'-UTR中的一段连续核苷酸对应的一段连续核苷酸，其代表基因的转录本的全长3'-UTR的至少5％、至少10％、至少20％、至少30％、至少40％、至少50％|、至少60％、至少70％、至少80％或至少90％。
术语“变体”是指天然存在的基因的转录本的3'-UTR的变体，优选是指病毒基因的转录本的3'-UTR的变体，更优选是指哺乳动物基因的转录本的3'-UTR的变体。所述变体可以是基因的转录本的修饰的3'-UTR。例如，与变体源自的天然存在的3'-UTR相比， 3'-UTR的变体可以呈现截短，末端延长，或者一个或多个核苷酸缺失、添加和/或突变。优选地，基因的转录本的3'-UTR的变体与变体源自的天然存在的3'-UTR有至少40％，优选至少50％，更优选至少60％，更优选至少70％，甚至更优选至少80％，甚至更优选至少90％，最优选至少95％相同。
如本文所用，术语“末端延长”指与变体源自的天然存在或修饰的3'-UTR相比，3'-UTR的变体在其N端或C端有一个或多个核苷酸的添加。
在一实施方案中，所述3'-UTR元件呈现至少约3个核苷酸，优选至少约5个核苷酸，更优选至少约10、15、20、25或30个核苷酸，甚至更优选至少约50个核苷酸，最优选至少约90个核苷酸的长度。在优选的实施方案中，所述3'-UTR元件呈现3至约500个核苷酸，优选5至约250个核苷酸，更优选90至215个核苷酸的长度。
在一实施方案中，所述核酸序列源自以下病毒基因的转录本的3'-UTR的核酸序列：HCV、CoV2、DENV2、TCV、CYBA、BYDA和CVB3，并且是截短的。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:8、19、21或23的核酸序列。
在一实施方案中，所述核酸序列源自小鼠基因AES的转录本的3'-UTR的核酸序列，并且是截短的。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:9的核酸序列。
在一实施方案中，所述核酸序列源自人基因AAT或AES的转录本的3'-UTR的核酸序列，并且是截短的。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:12或96的核酸序列。
在一实施方案中，所述核酸序列源自牛基因AES的转录本的3'-UTR的核酸序列，并且是截短的。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:98的核酸序列。
在一实施方案中，所述核酸序列源自以下病毒基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、DENV2、TCV、CYBA、BYDA和CVB3，其中所述变体与所源自的核酸序列相比，其包含截短和1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:6、7、20、22、30、31或32的核酸序列。
在一实施方案中，所述核酸序列源自牛基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短和1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:94的核酸序列。
在一实施方案中，所述核酸序列源自小鼠基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短和1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:97的核酸序列。
在一实施方案中，所述核酸序列源自小鼠基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短和末端延长。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:9的核酸序列，并且还包含SEQ ID NO:92或12的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:10、13或28的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:9的核酸序列，并且还包含SEQ ID NO:92和12的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:33的核酸序列。
在一实施方案中，所述核酸序列源自人基因AAT的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短和末端延长。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:12的核酸序列，并且还包含SEQ ID NO:6、9、92、94、97或98的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:11、13、14、28、34、35或36的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:12的核酸序列，并且还包含SEQ ID NO:9和92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:33的核酸序列。
在一实施方案中，所述核酸序列源自牛基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短和末端延长。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:98的核酸序列，并且还包含SEQ ID NO:12的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:35的核酸序列。
在一实施方案中，所述核酸序列源自牛基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:94的核酸序列，并且还包含SEQ ID NO:12的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:11的核酸序列。
在一实施方案中，所述核酸序列源自以下病毒基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、DENV2、TCV、CYBA、BYDA和CVB3，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:6的核酸序列，并且还包含SEQ ID NO:92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:15的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:7的核酸序列，并且还包含SEQ ID NO:92的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:24的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:6的核酸序列，并且还包含SEQ ID NO:12的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:36的核酸序列。
在一实施方案中，所述核酸序列源自小鼠基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:97的核酸序列，并且还包含SEQ ID NO:12的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:34的核酸序列。
在一实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含SEQ ID NO:6、7、8、9、10、11、12、13、14、15、19、20、21、22、23、24、28、30、31、32、33、34、35、36、94、96、97或98的核酸序列。
在一优选实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含源自以下基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、CVB3、AES和AAT，所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一优选实施方案中，所述核酸序列源自以下病毒基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2和CVB3，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一优选实施方案中，所述核酸序列源自小鼠基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一优选实施方案中，所述核酸序列源自人基因AAT的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一优选实施方案中，所述核酸序列源自牛基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一优选的实施方案中，所述核酸序列源自以下病毒基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2和CVB3；或源自小鼠基因AES的转录本的3'-UTR的核酸序列或其变体；或源自人基因AAT的转录本的3'-UTR的核酸序列或其变体；或源自牛基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:6、7、8、9、10、11、12、13、14或15的核酸序列。
在一实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含SEQ ID NO:16、42或43的核酸序列。
在一优选实施方案中，所述3'-UTR元件包含SEQ ID NO:16的核酸序列。
在一实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含选自以下的核酸序列：
(i)其中所述3'-UTR元件包含SEQ ID NO:44所示核酸序列的变体，所述变体与SEQ ID NO:44所示核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
(ii)其中所述3'-UTR元件包含源自以下基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、DENV2、TCV、CYBA、BYDA、CVB3、AES和AAT，所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
(iii)其中所述3'-UTR元件包含SEQ ID NO:16、42或43的核酸序列。
在一实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含选自以下的核酸序列：
SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42或43。
具体3'-UTR元件的核酸序列见表1。
表1. 3'-UTR元件的核酸序列
Figure PCTCN2022131289-appb-000001
Figure PCTCN2022131289-appb-000002
Figure PCTCN2022131289-appb-000003
在一优选的实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含选自以下的核酸序列：
(i)其中所述3'-UTR元件包含SEQ ID NO:44所示核酸序列的变体，所述变体与SEQ ID NO:44所示核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
(ii)其中所述3'-UTR元件包含源自以下基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、CVB3、AES和AAT，所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
(iii)其中所述3'-UTR元件包含SEQ ID NO:16的核酸序列。
在一特别优选的实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含选自以下的核酸 序列：
(i)其中所述3'-UTR元件包含SEQ ID NO:44所示核酸序列的变体，所述变体与SEQ ID NO:44所示核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
(ii)其中所述3'-UTR元件包含源自以下基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、CVB3、AES和AAT，所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
(iii)其中所述3'-UTR元件包含SEQ ID NO:16的核酸序列；
或者上述核酸序列的相应RNA序列。
在一具体的实施方案中，所述人工核酸分子，其包含
a.至少一个开放阅读框(ORF)；和
b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含选自以下的核酸序列：
SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或16。
如本文所用，术语“5'-非翻译区元件(5'-UTR元件)”是指人工核酸分子的一部分，其位于开放阅读框的5'端(即“上游”)，并且其不翻译为蛋白。通常，5'-UTR在转录起始位点起始并且在开放阅读框的起始密码子之前的一个核苷酸处终止。5'-UTR在调节基因表达中起关键作用。5'-UTR的DNA序列中可以包含许多顺式调节元件，它们可以与转录机制相互作用以调节信使RNA(mRNA)的丰度。此外，转录的5'-UTR由多种基于RNA的调控元件组成，包括5'帽结构、二级结构、RNA结合蛋白基序、上游开放阅读框(uORF)、内部核糖体进入位点、末端寡嘧啶(TOP)束和G-四链体。这些元素可以改变mRNA翻译的效率；有些还可以通过稳定性或降解的变化来影响mRNA转录水平(参见，例如Lim,Y.,et al.Multiplexed functional genomic analysis of 5'untranslated region mutations across the spectrum of prostate cancer.Nat Commun 12,4217(2021))。
在一优选的实施方案中，所述人工核酸分子其还包含至少一个5'-非翻译区元件(5'-UTR元件)。
在一优选的实施方案中，所述5'-非翻译区元件(5'-UTR元件)包含SEQ ID NO:45的核酸序列。
如本文所用，术语“poly(A)序列”或“poly(A)尾”是指包含连续或不连续腺苷酸的核苷酸序列。poly(A)序列通常位于RNA的3'端，例如3'-UTR的3'端(下游)。在一些实施方案中，poly(A)序列在其3'端不包含腺苷酸以外的核苷酸。Poly(A)序列可以在制备IVT-RNA期间，由DNA依赖性RNA聚合酶根据DNA模板的编码序列转录产生，或者通过不依赖于DNA的RNA聚合酶(poly(A)聚合酶)连接至IVT-RNA的游离3'端，例如3'-UTR的3'端。在一实施方案中，所述人工核酸分子还包含聚腺苷酸序列。
如本文所用，术语“聚胞苷酸序列”是指包含连续或不连续胞苷酸的核苷酸序列。聚 胞苷酸序列通常位于RNA的3'端，例如3'-UTR的3'端(下游)。在一些实施方案中，聚胞苷酸序列在其3'端不包含胞苷酸以外的核苷酸。在一实施方案中，所述人工核酸分子还包含聚胞苷酸序列。
如本文所用，术语“5'帽”一般涉及通过5'至5'三磷酸键连接至mRNA的5'端的N7-甲基鸟苷结构(又称为“m7G帽”、“m7Gppp-”)。5'帽可以在体外转录中共转录加至RNA中(例如使用抗反向帽类似物“ARCA”)，或者可以利用加帽酶在转录后连接至RNA。在一实施方案中，所述人工核酸分子还包含5'帽结构。
如本文所用，“茎环”(不管其是组蛋白茎环还是不是组蛋白茎环)通常可以存在于单链DNA中，或者更常见地存在于RNA中。该结构还称为发夹或发夹环，并且通常由连续序列内的茎和(末端)环组成，其中所述茎由两条由在一定程度上作为间隔体的短序列隔开的相邻的完全或部分反向互补的序列形成，所述作为间隔体的短序列成为所述茎环结构的环。这两条相邻的完全或部分反向互补的序列可以定义为，例如，茎环元件茎1和茎2。当这两条相邻的完全或部分反向互补的序列，例如，茎环元件茎1和茎2，彼此形成碱基配对时，形成双链的核酸序列片段，其在其末端包含由位于连续序列上的茎环元件茎1和茎2之间的短序列形成的不配对的环，从而形成茎环。
如本文所用，“组蛋白茎环”来源于组蛋白基因(例如，来源于组蛋白家族H1，H2A，H2B，H3，H4的基因)，并且包含两条相邻的完全或部分反向互补的序列的分子内碱基配对，由此形成茎环。在一实施方案中，所述人工核酸分子还包含组蛋白茎环。
在一实施方案中，所述人工核酸分子还包含5'帽结构、聚胞苷酸序列、聚腺苷酸序列或组蛋白茎环。
在一实施方案中，所述人工核酸分子还包含5'帽结构和聚胞苷酸序列。
在一实施方案中，所述人工核酸分子还包含5'帽结构和聚腺苷酸序列。
在一实施方案中，所述人工核酸分子还包含5'帽结构、聚胞苷酸序列和组蛋白茎环。
在一实施方案中，所述人工核酸分子还包含5'帽结构、聚腺苷酸序列和组蛋白茎环。
在一实施方案中，所述人工核酸分子还包含5'帽结构、聚胞苷酸序列、聚腺苷酸序列和组蛋白茎环。
在一优选的实施方案中，所述人工核酸分子包含聚腺苷酸序列，所述聚腺苷酸序列包含SEQ ID NO:46的核酸序列。
本文定义的人工核酸分子可以使用本领域已知的任意方法制备，包括合成法，例如，固相合成，以及体外方法，例如体外转录反应或体内反应，例如DNA质粒在细菌中的体内繁殖。本发明的人工核酸分子可以针对用于表达的宿主细胞进行密码子优化。
在一优选的实施方案中，所述开放阅读框(ORF)是经过密码子优化的。
在一实施方案中，所述人工核酸分子是RNA。
在一优选的实施方案中，所述人工核酸分子是mRNA。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88或89的核酸序列。
在一实施方案中，所述3'-UTR元件包含SEQ ID NO:47、48、49、50、51、52、53、54、55、56、57、58、59、60、61或62的核酸序列。
经过修饰的核苷酸
在一些方式中，所述的mRNA包括经过修饰的核苷酸，其中修饰的核苷酸选择如下一种或者几种核苷酸：2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、C-5丙炔基-胞苷、C-5丙炔基-尿苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷、C5-甲基胞苷、2-氨基腺苷、7-脱氮腺苷、7-脱氮鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟嘌呤、假尿苷、N-1-甲基-假尿苷、2-硫代尿苷以及2-硫代胞苷；甲基化碱基；插入碱基；2'-氟代核糖、核糖、2'-脱氧核糖、阿拉伯糖以及己糖；硫代磷酸基和5'-N-亚磷酰胺键。以及PCT/CN2020/074825，PCT/CN2020/106696中所描述的改性核苷酸进行修饰。
载体和宿主细胞
在又一方面，本发明还提供了包含本发明的人工核酸分子的表达载体。表达载体可以进一步包含额外的核酸序列，例如调控序列和抗生素抗性基因。本发明的人工核酸分子可以存在于一种或多种表达载体中。
在一实施方案中，所述载体是DNA载体。
在一实施方案中，所述载体是质粒载体或病毒载体。
在一优选的实施方案中，所述载体是质粒载体。例如pUC57质粒载体。
在一实施方案中，所述载体是环形分子。
在一实施方案中，本发明的人工核酸分子制备为重组核酸。可使用本领域众所周知的技术制备重组核酸，例如化学合成、DNA重组技术(例如聚合酶链式反应(PCR)技术)等。
在又一方面，本发明还提供了一种产生本发明的人工核酸分子的方法，所述方法包括：
a)合成本发明的人工核酸分子；或者
b)通过本发明的载体合成人工核酸分子。
在又一方面，本发明还提供一种用于增加人工核酸分子、优选mRNA分子或载体的翻译效率的方法，所述方法包括连接开放阅读框与本发明所定义的3'-UTR元件。
本发明还提供了一种细胞，其包含本发明的人工核酸分子或载体。可以采用本领域已知的各种方法将本发明的人工核酸分子或载体导入合适的细胞中。这类方法包括但不限于脂质体转染、电穿孔、病毒转导和磷酸钙转染等。
在优选的实施方案中，细胞用于表达本发明的人工核酸分子中的ORF所编码的肽或蛋白。细胞的实例包括但不限于原核细胞(例如细菌，例如大肠杆菌)和真核细胞(例如酵母、昆虫细胞、哺乳动物细胞)。合适的哺乳动物宿主细胞包括但不限于外人宫颈癌细胞(HeLa细胞)、人类胚胎肾细胞(HEK细胞，例如HEK 293细胞)、中国仓鼠卵巢(CHO)细胞和其他哺乳动物细胞。
在一实施方案中，所述细胞是哺乳动物细胞。
在一优选的实施方案中，所述细胞是人受试者分离的细胞。
脂质组合物
在又一方面，本发明还提供一种脂质组合物。所述脂质组合物为脂质递送载体，脂质可将本发明的人工核酸分子包封形成纳米颗粒，从而递送至生物体内。
如本文所用，术语“脂质”是指包含疏水部分并且任选地还包含亲水部分的有机化合物。脂质通常难溶于水但可溶于许多有机溶剂。通常，包含疏水部分和亲水部分的两亲性脂质可以在水环境中组织为脂质双层结构，例如以囊泡形式存在。脂质可以包括但不限于：脂肪酸、甘油酯、磷脂、鞘脂、糖脂和类固醇和胆固醇酯等。
如本文所用，“脂质纳米颗粒”或“LNP”是指一种具有均匀脂质核心的脂质囊泡，其是由脂质形成的颗粒，脂质成分发生分子间相互作用而形成纳米结构实体。核酸(例如mRNA)被包封在脂质中。
特别优选的脂质组合物可以是例如本文所述的脂质多聚复合物(LPP)。制备这类组合物的方法可以如本文所述。LPP是具有核-壳结构的颗粒，其中核酸包含于多聚复合物中，而多聚复合物本身被包封于生物相容性脂质双层壳中以构成本发明的脂质纳米颗粒。在一些实施方案中，本发明的脂质组合物为脂质多聚复合物(LPP)。在一些实施方案中，本发明的脂质组合物为包含人工核酸分子的脂质多聚复合物(LPP)。
在一些实施方案中，所述的包封本发明的人工核酸分子的脂质选择如下脂质的一种或者几种：阳离子脂质、磷脂、类固醇和/或聚乙二醇修饰的脂质。在一优选实施方案中，所述阳离子脂质为可离子化阳离子脂质。
本发明的脂质组合物，其包含本发明的人工核酸分子以及包封所述人工核酸分子的脂质。所述包封人工核酸分子的脂质包含阳离子脂质、磷脂、类固醇和聚乙二醇修饰的脂质。
在一实施方案中，脂质组合物包含阳离子脂质，其中阳离子脂质包含DOTMA、DOTAP、DDAB、DOSPA、DODAC、DODAP、DC-Chol、DMRIE、DMOBA、DLinDMA、DLenDMA、CLinDMA、DMORIE、DLDMA、DMDMA、DOGS、N4-胆固醇基-精胺、 DLin-KC2-DMA、DLin-MC3-DMA、如本文所述的式(I)、(II)、(III)或(IV)的化合物或其组合。在一优选实施方案中，阳离子脂质包含M5、MC3、ALC-0315、SM-102。在一优选实施方案中，阳离子脂质包含SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2。在一优选实施方案中，阳离子脂质包含M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2。
在一实施方案中，脂质组合物包含磷脂和/或类固醇。在一实施方案中，脂质组合物包含如本文所述的磷脂，其中磷脂包含1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-双十一烷酰基-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0Diether PC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME 16.0PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺(LPE)或其组合。在一实施方案中，脂质组合物包含如本文所述的类固醇，其中类固醇包含胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚及其衍生物。在一实施方案中，脂质组合物包含如本文所述的磷脂和类固醇。在一实施方案中，脂质组合物包含DOPE。在一实施方案中，脂质组合物包含DSPC。在一实施方案中，脂质组合物包含胆固醇。在一实施方案中，脂质组合物包含DOPE和胆固醇。在一实施方案中，脂质组合物包含DSPC和胆固醇。
在一实施方案中，脂质组合物包含阳离子脂质M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2，磷脂DOPE和胆固醇。在一实施方案中，脂质组合物包含阳离子脂质M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2，磷脂DSPC和胆固醇。
在一些实施方案中，包封本发明的人工核酸分子的脂质进一步包含聚乙二醇修饰的脂质。在一实施方案中，聚乙二醇修饰的脂质包含DMG-PEG(例如DMG-PEG 2000)、DOG-PEG和DSPE-PEG或其组合。在一实施方案中，聚乙二醇修饰的脂质为DSPE-PEG。在一实施方案中，聚乙二醇修饰的脂质为DMG-PEG(例如DMG-PEG 2000)。
在一实施方案中，脂质组合物包含阳离子脂质、DOPE、胆固醇和DSPE-PEG。
在一实施方案中，脂质组合物包含阳离子脂质、DSPC、胆固醇和DSPE-PEG。
在一实施方案中，脂质组合物包含阳离子脂质、DSPC、胆固醇和DMG-PEG。
在一优选的实施方案中，脂质组合物包含阳离子脂质、DOPE、胆固醇和DMG-PEG。
在一优选的实施方案中，脂质组合物包含阳离子脂质M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2、DOPE、胆固醇和DMG-PEG。
在一些实施方案中，本发明的脂质组合物进一步包含阳离子聚合物，所述阳离子聚合物与所述人工核酸分子缔合为复合物，共同包封在所述脂质中。
在一实施方案中，阳离子聚合物包含聚-L-赖氨酸、鱼精蛋白、聚乙烯亚胺(PEI)或其组合。在一实施方案中，阳离子聚合物为鱼精蛋白。在一实施方案中，阳离子聚合物为聚乙烯亚胺。
在一实施方案中，脂质组合物中脂质的量以摩尔百分比(摩尔％)来计算，所述摩尔百分比基于组合物中脂质的总摩尔来确定。
在一实施方案中，脂质组合物中阳离子脂质的量为约10-约70摩尔％。在一些实施方案中，脂质组合物中阳离子脂质的量为约20-约60摩尔％、约30-约50摩尔％、约35-约50摩尔％、约35-约45摩尔％、约38-约45摩尔％、约40-约45摩尔％、约40-约50摩尔％或约45-约50摩尔％。
在一实施方案中，脂质组合物中磷脂的量为约10-约70摩尔％。在一实施方案中，脂质组合物中磷脂的量为约20-约60摩尔％、约30-约50摩尔％、约10-约30摩尔％、约10-约20摩尔％或约10-约15摩尔％。
在一实施方案中，脂质组合物中胆固醇的量为约10-约70摩尔％。在一实施方案中，脂质组合物中胆固醇的量为约20-约60摩尔％、约24-44摩尔％、约30-约50摩尔％、约35-约40摩尔％、约35-约45摩尔％、约40-约45摩尔％或约45-约50摩尔％。
在一实施方案中，脂质组合物中聚乙二醇修饰的脂质的量为约0.05-约20摩尔％。在一实施方案中，脂质组合物中聚乙二醇修饰的脂质的量为约0.5-约15摩尔％、约1-约10摩尔％、约5-约15摩尔％、约1-约5摩尔％、约1-约1.5摩尔％、约1.5-约3摩尔％或约2-5摩尔％。
在一实施方案中，脂质组合物包含10-70摩尔％的阳离子脂质、10-70摩尔％的磷脂、10-70摩尔％的类固醇和0.05-20摩尔％的聚乙二醇修饰的脂质。在一优选实施方案中，脂质组合物包含35-50摩尔％的阳离子脂质、10-30摩尔％的磷脂、24-44摩尔％的类固 醇和1-1.5摩尔％的聚乙二醇修饰的脂质。
在一实施方案中，LPP包含本发明的人工核酸分子以及包封所述人工核酸分子的脂质，其中所述包封人工核酸分子的脂质包含阳离子脂质、磷脂、类固醇和聚乙二醇修饰的脂质；所述LPP还包含阳离子聚合物，其中所述阳离子聚合物与所述人工核酸分子缔合为复合物。在一实施方案中，本发明的脂质组合物，其包含本发明的人工核酸分子以及包封所述人工核酸分子的脂质，其中所述包封人工核酸分子的脂质包含阳离子脂质、磷脂、类固醇和聚乙二醇修饰的脂质；所述脂质组合物还包含阳离子聚合物，其中所述阳离子聚合物与所述人工核酸分子缔合为复合物，共同包封在所述脂质中形成脂质多聚复合物。在一实施方案中中，所述脂质组合物包含2.5-20摩尔％的聚乙二醇修饰的脂质，基于所述脂质组合物中所有脂质的总量。在一实施方案中，所述磷脂选自1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、二硬脂酰基磷脂酰胆碱(DSPC)或其组合。在一实施方案中，所述类固醇为胆固醇。在一实施方案中，所述阳离子聚合物为鱼精蛋白。在一实施方案中，所述聚乙二醇修饰的脂质选自1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)、1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-聚(乙二醇)(DSPE-PEG)或其组合。在一实施方案中，所述阳离子脂质选自M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2。
在一实施方案中，所述包封复合物的脂质包含50摩尔％的M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2，10摩尔％的DOPE，38.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG。在一实施方案中，所述包封复合物的脂质包含40摩尔％的M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2，15摩尔％的DOPE，43.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG。
阳离子脂质
阳离子脂质是在指定pH下可以带有净正电荷的脂质。带有净正电荷的脂质可以通过静电相互作用与核酸缔合。
阳离子脂质的实例包括但不限于1,2-二-O-十八烯基-3-三甲基铵丙烷(1,2-di-O-octadecenyl-3-trimethylammonium-propane，DOTMA)、1,2-二油酰基-3-三甲基铵-丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、双十烷基二甲基溴化铵(Didecyldimethylammonium bromide，DDAB)、2,3-二油酰基氧基-N-[2(精胺羧酰胺)乙基]-N,N-二甲基-l-丙胺鎓三氟乙酸盐(2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate，DOSPA)、双十八烷基二甲基氯化铵(dioctadecyldimethyl ammonium chloride，DODAC)、1,2-二油酰基-3-二甲基铵-丙烷(1,2-dioleoyl-3-dimethylammonium-propane，DODAP)、3-(N—(N′,N′-二甲基氨基乙烷)-氨甲酰基)胆固醇(3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol， DC-Chol)、2,3-二(十四烷基氧基)丙基-(2-羟基乙基)-二甲基氨鎓(2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium，DMRIE)、N,N-二甲基-3,4-二油基氧基苄胺(N,N-dimethyl-3,4-dioleyloxybenzylamine，DMOBA)、1,2-二亚油基氧基-N,N-二甲基氨基丙烷(1,2-dilinoleyloxy-N,N-dimethylaminopropane，DLinDMA)、1,2-二亚油烯基氧基-N,N-二甲基氨基丙烷(1,2-dilinolenyloxy-N,N-dimethylaminopropane，DLenDMA)、3-二甲基氨基-2-(胆甾-5-烯-3-β-氧基丁烷-4-氧基)-1-(顺式,顺式-9,12-十八碳二烯基氧基)丙烷(3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane，CLinDMA)、N-(2-氨基乙基)-N,N-二甲基-2,3-双(十四烷基氧基)丙烷-1-胺鎓溴化物(N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide，DMORIE)、N,N-二甲基-2,3-双(十二烷基氧基)丙烷-1-胺(N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine，DLDMA)、N,N-二甲基-2,3-双(十四烷基氧基)丙烷-1-胺(N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine，DMDMA)、双十八烷基酰氨基甘氨酰基精胺(dioctadecylamidoglycyl spermine，DOGS)、N4-胆固醇基-精胺(N4-cholesteryl-spermine)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧戊环(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane，DLin-KC2-DMA)、三十七烷基-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate，DLin-MC3-DMA)、如本文所述的式(I)、(II)、(III)或(IV)的化合物或其组合。
在一些实施方案中，阳离子脂质优选为可离子化阳离子脂质。可离子化阳离子脂质在例如酸性pH下带有净正电荷，而在较高pH(例如生理pH)下是中性的。可离子化阳离子脂质的实例包括但不限于：双十八烷基酰氨基甘氨酰基精胺(dioctadecylamidoglycyl spermine，DOGS)、N4-胆固醇基-精胺(N4-cholesteryl-spermine)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧戊环(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane，DLin-KC2-DMA)、三十七烷基-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate，DLin-MC3-DMA)、如本文所述的式(I)、(II)、(III)或(IV)的化合物或其组合。
在一实施方案中，阳离子脂质包含式(I)的化合物或其药学上可接受的盐：
Figure PCTCN2022131289-appb-000004
其中，
R 1和R 2各自独立选自键、C 1-C 12烷基和C 2-C 12烯基；
R 3和R 4各自独立选自C 1-C 12烷基、C 2-C 12烯基、C 6-C 10芳基和5-10元杂芳基；并且R 3和R 4各自独立任选被t个R 6取代，t为选自1-5的整数；
R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基；
M 1和M 2各自独立地选自键、H、-O-、-S-、-C(O)-、-OC(O)-、-C(O)O-、-OC(O)O-、-SC(S)-、-C(S)S-、3-10元杂环、-NR 7-，或者
R 5与M 1和M 2之一连同所连接的N原子一起形成3-10元杂环，且对应的R 1/R 3或
者R 2/R 4不存在，所述杂环任选地被R 7取代；
R 5选自C 3-8碳环、-C 1-12亚烷基-Q，Q选自H、-OR 7、-SR 7、-OC(O)R 7、-C(O)OR 7、-N(R 7)C(O)R 7、-N(R 7)S(O) 2R 7、-N(R 7)C(S)R 7、-N(R 7) 2、氰基、C 3-8碳环、3-10元杂环、C 6-C 10芳基，上述基团各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代；
m和n各自独立为选自0-12的整数；
所述烷基、烯基和亚烷基各自任选地独立地被一个或多个选自以下的基团中断：-O-、-S-、-NR 7-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C 3-8碳环，且所述烷基、烯基和亚烷基各自任选地被一个或多个R 7取代；
R 7各自独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、C 3-8碳环，上述基团各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代。
在一实施方案中，R 1和R 2各自独立选自C 1-C 12烷基和C 2-C 12烯基，例如C 1-C 12烷基。在又一实施方案中，R 1和R 2之一为键，另一个独立地选自C 1-C 12烷基和C 2-C 12烯基，例如C 1-C 12烷基。
在一实施方案中，R 3和R 4各自独立选自C 1-C 12烷基、C 2-C 12烯基、C 6-C 10芳基和5-10元杂芳基。在又一实施方案中，R 3和R 4各自独立选自C 1-C 12烷基和C 2-C 12烯基。
R 3和R 4可以各自独立任选被t个R 6取代，t为1、2、3、4、5。在一实施方案中，R 6各自独立选自C 1-C 12烷基。
在又一实施方案中，R 3和R 4中至少一个为C 6-C 10芳基或5-10元杂芳基，例如C 6-C 10芳基。
在一实施方案中，R 5选自C 3-8碳环、-C 1-12亚烷基-Q。Q可以选自H、-OR 7、-SR 7、-OC(O)R 7、-C(O)OR 7、-N(R 7)C(O)R 7、-N(R 7)S(O) 2R 7、-N(R 7)C(S)R 7、-N(R 7) 2、氰基、C 3-8碳环、3-10元杂环、C 6-C 10芳基。上述基团，包括涵盖Q的选项的基团，当合适时，可以各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代。
在又一实施方案中，R 5选自C 3-8碳环、-C 1-12亚烷基-Q，Q选自H、-OR 7、-SR 7、-OC(O)R 7、-C(O)OR 7、-N(R 7)C(O)R 7、-N(R 7)S(O) 2R 7、-N(R 7)C(S)R 7、-N(R 7) 2、氰基、 C 3-8碳环、3-10元杂环、C 6-C 10芳基。上述基团，包括涵盖Q的选项的基团，当合适时，可以各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代。
在式(I)的化合物中，R 7可以各自独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、C 3-8碳环，优选选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基和5-10元杂芳基。上述基团(当合适时，例如H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、C 3-8碳环)各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代。
在一实施方案中，上述描述中的各个基团，例如C 3-8碳环、-C 1-12亚烷基-Q，包括涵盖Q选项的-OR 7、-SR 7、-OC(O)R 7、-C(O)OR 7、-N(R 7)C(O)R 7、-N(R 7)S(O) 2R 7、-N(R 7)C(S)R 7、-N(R 7) 2、C 3-8碳环、3-10元杂环、C 6-C 10芳基，C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、C 3-8碳环等可以各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代。
在一实施方案中，式(I)化合物中的(例如R 1-R 7中提及的)烷基、烯基和亚烷基可以各自任选地独立地被一个或多个选自以下的基团中断：-O-、-S-、-NR 7-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C 3-8碳环，且所述烷基、烯基和亚烷基各自任选地被一个或多个R 7取代。即，所述烷基、烯基和亚烷基的链(直链或支链)中，可以各自任选地包含一个或多个选自以下的基团：-O-、-S-、-NR 7-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C 3-8碳环。
R 7各自独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、C 3-8碳环；优选地，R 7独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基和5-10元杂芳基。上述基团(当合适时，例如H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、C 3-8碳环)各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代。
在式(I)的化合物中，m和n可以各自独立为选自0-12的整数，例如，0、1、2、3、4、5、6、7、8、9、10、11、12。当取0时，表示对应的基团不存在。
在一实施方案中，M 1或M 2为键，相应的m或n不为0，M 1或M 2前的碳链与相应的R 1或R 2连接。
在一实施方案中，m或n为0，相应的M 1或M 2不为键，N原子直接与M 1或M 2相连接。
在一实施方案中M 1或M 2为键，相应的m或n为0，N原子直接与相应的R 1或R 2相连接。
在一实施方案中，M 1和M 2各自独立地选自-C(O)-、-OC(O)-、-C(O)O-和-OC(O)O-。在又一实施方案中，M 1和M 2各自独立地选自-NR 7-，R 7如上文所述。
在另一实施方案中，R 5与M 1和M 2之一连同所连接的N原子一起形成3-10元杂环，且对应的R 1/R 3或者R 2/R 4不存在，所述杂环任选地被R 7取代，R 7如上文所述。
在一实施方案中，R 5选自-C 1-12亚烷基-Q，Q选自H、-OR 7、-OC(O)R 7、-C(O)OR 7、-N(R 7)C(O)R 7、-N(R 7) 2、氰基，R 7如上文所述。
在一优选实施方案中，R 1和R 2各自独立选自C 1-C 12烷基和C 2-C 12烯基；
其中R 3和R 4各自独立选自C 1-C 12烷基和C 2-C 12烯基；并且R 3和R 4各自独立任选被t个R 6取代，t为选自1-5的整数；R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基。
M 1和M 2各自独立选自-OC(O)-、-C(O)O-、-OC(O)O-、-SC(S)-和-C(S)S-；
R 5选自-C 1- 12亚烷基-Q，Q选自-OR 7和-SR 7，R 7独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基和5-10元杂芳基；
m和n各自独立为选自1-12的整数。
在一优选实施方案中，阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐：
Figure PCTCN2022131289-appb-000005
Figure PCTCN2022131289-appb-000006
在一优选实施方案中，阳离子脂质包含M5或SM-102。
在一优选实施方案中，阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐：
Figure PCTCN2022131289-appb-000007
Figure PCTCN2022131289-appb-000008
在一优选实施方案中，阳离子脂质包含MC3。
在一优选实施方案中，阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐：
Figure PCTCN2022131289-appb-000009
在一优选实施方案中，阳离子脂质包含ALC-0315。
在一实施方案中，阳离子脂质包含式(I)的化合物或其药学上可接受的盐：
Figure PCTCN2022131289-appb-000010
R 1和R 2各自独立选自C 1-C 12烷基和C 2-C 12烯基；
R 3和R 4各自独立选自C 1-C 12烷基、C 2-C 12烯基、C 6-C 10芳基和5-10元杂芳基；
条件是R 3和R 4中至少一个为C 6-C 10芳基或5-10元杂芳基，并且R 3和R 4各自独立任选被t个R 6取代，t为选自1-5的整数；R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基；
M 1和M 2各自独立选自-OC(O)-、-C(O)O-、-OC(O)O-、-SC(S)-和-C(S)S-；
R 5选自-C 1-12亚烷基-Q，Q选自-OR 7和-SR 7，R 7独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基和5-10元杂芳基；
m和n各自独立为选自1-12的整数。
在一实施方案中，R 2选自C 1-C 12烷基。在另一实施方案中，R 2选自C 1-C 6烷基。
在一实施方案中，R 3和R 4之一为C 6-C 10芳基或5-10元杂芳基，另一个为C 1-C 12烷基或C 2-C 12烯基。
在一具体实施方案中，R 3和R 4各自独立选自C 1-C 12烷基和苯基，条件是R 3和R 4中至少一个为苯基。在另一实施方案中，R 3和R 4之一为苯基，另一个为C 1-C 12烷基。
在又一实施方案中，R 3和R 4各自独立被t个R 6取代，t为选自1-5的整数；例如1、2、3、4或5。优选地，t为1-3的整数，例如1、2或3，特别是1或2。
在一实施方案中，R 6各自独立选自C 1-C 12烷基，例如C 1-C 10烷基。
在一实施方案中，t为1，R 6取代于苯环上相对于R 1或R 2的间位或对位。
在另一实施方案中，t为2，R 6取代于苯环上相对于R 1或R 2的间位和对位。
在一实施方案中，R 4取代于R 2的1位或末位。所述1位是指R 2中与M 2直接相连的C原子的位置。所述末位是指R 2中与M 2距离最远的C原子的位置。在一具体实施方案中，R 4选自C 1-C 12烷基，R 3为苯基。
在一实施方案中，R 3取代于R 1的1位或末位。所述1位是指R 1中与M 1直接相连的C原子的位置。所述末位是指R 1中与M 1距离最远的C原子的位置。在一具体实施方案中，R 3选自C 1-C 12烷基，R 4为苯基。
在一实施方案中，M 1和M 2各自独立选自-OC(O)-、-C(O)O-和-OC(O)O-。
在一实施方案中，R 5选自-C 1-5亚烷基-Q，例如C 1、C 2、C 3、C 4或C 5亚烷基-Q。在示例性实施方案中，R 5选自-C 1-3亚烷基-Q，例如C 1、C 2或C 3亚烷基-Q。
在另一实施方案中，Q选自-OH和-SH，特别是-OH。
在一些实施方案中，m和n各自独立为选自2-9的整数，例如2、3、4、5、6、7、8或9。优选地，m和n各自独立为选自2-7的整数，例如2、3、4、5、6或7，更优选地，m和n各自独立为选自5-7的整数，例如5、6或7。
在某些实施方案中，式(I)的化合物包括式(II)所示的化合物：
Figure PCTCN2022131289-appb-000011
或其药物可接受的盐，其中各基团如本文所定义。
在一实施方案中，
R 1选自C 1-C 6烷基；
R 2选自C 1-C 10烷基；
R 4选自C 1-C 10烷基；
M 1和M 2各自独立选自-OC(O)-、-C(O)O-和-OC(O)O-；
R 5选自-C 1-5亚烷基-Q，Q选自-OR 7和-SR 7，R 7独立选自H、C 1-C 12烷基和C 2-C 12烯基；
R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基，特别是C 1-C 12烷基；
m和n各自独立为选自2-9的整数，例如2、3、4、5、6、7、8或9；
t为选自1-3的整数。
在一实施方案中，R 5选自-C 1-3亚烷基-Q，Q选自-OH和-SH，特别是-OH。
在一实施方案中，m和n各自独立为选自2-7的整数，例如2、3、4、5、6或7。
在一些实施方案中，t为1或2。
在一实施方案中，R 4取代于R 2的1位或末位。所述1位是指R 2中与M 2直接相连的C原子的位置。所述末位是指R 2中与M 2距离最远的C原子的位置。
在一实施方案中，t为1，R 6取代于苯环上相对于R 1的间位或对位。
在另一实施方案中，t为2，R 6取代于苯环上相对于R 1的间位和对位。
在某些实施方案中，式(I)的化合物包括式(III)所示的化合物：
Figure PCTCN2022131289-appb-000012
或其药物可接受的盐，其中各基团如本文所定义。
在一实施方案中，
R 1选自C 1-C 6烷基；
R 2选自C 1-C 10烷基；
R 4选自C 1-C 10烷基；
R 5选自-C 1-3亚烷基-Q，Q选自-OH和-SH，特别是-OH；
t为1或2；
R 6选自C 1-C 12烷基和C 2-C 12烯基，特别是C 1-C 12烷基；
m和n各自独立为选自2-7的整数，例如2、3、4、5、6或7。
在一实施方案中，R 4取代于R 2的1位或末位。所述1位是指R 2中与
Figure PCTCN2022131289-appb-000013
部分直接相连的C原子的位置。所述末位是指R 2中与
Figure PCTCN2022131289-appb-000014
部分距离最远的C原子的位置。
在一实施方案中，t为1，R 6取代于苯环上相对于R 1的间位或对位。
在另一实施方案中，t为2，R 6取代于苯环上相对于R 1的间位和对位。
在某些实施方案中，式(I)的化合物包括式(IV)所示的化合物：
Figure PCTCN2022131289-appb-000015
或其药物可接受的盐，其中各基团如本文所定义。
在一实施方案中，
R 1选自C 1-C 6烷基；
R 2选自C 1-C 10烷基；
R 4选自C 1-C 10烷基；
t为1或2；
R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基，特别是C 1-C 12烷基；
m和n各自独立为选自2-7的整数，例如2、3、4、5、6或7。
在一实施方案中，R 4取代于R 2的1位或末位。所述1位是指R 2中与
Figure PCTCN2022131289-appb-000016
部分直接相连的C原子的位置。所述末位是指R 2中与
Figure PCTCN2022131289-appb-000017
部分距离最远的C原子的位置。
在一实施方案中，t为1，R 6取代于苯环上相对于R 1的间位或对位。
在另一实施方案中，t为2，R 6取代于苯环上相对于R 1的间位和对位。
在一特定的实施方案中，本发明的脂质化合物中的取代基中(例如，R 1-R 7)不包含烯基。
在优选的实施方案中，阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐：
Figure PCTCN2022131289-appb-000018
Figure PCTCN2022131289-appb-000019
Figure PCTCN2022131289-appb-000020
在一优选实施方案中，阳离子脂质包含以下脂质化合物：SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2。
在一优选实施方案中，阳离子脂质包含以下脂质化合物：M5、MC3、ALC-0315、SM-102、SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2。
磷脂
本发明的脂质组合物中包含磷脂，其可以辅助脂质组合物的细胞渗透。
磷脂的实例包括但不限于：1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-双十一烷酰基-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0Diether PC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME 16.0PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺(LPE)或其组合。
类固醇
本发明的脂质组合物中包含类固醇，其可以充当脂质组合物的结构组分。
类固醇的实例包括但不限于例如胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚及其衍生物。
聚乙二醇修饰的脂质
如本文所用，术语“聚乙二醇修饰的脂质”或“PEG改性的脂质”或“PEG脂质”指包含聚乙二醇部分和脂质部分的分子，其是用聚乙二醇改性的脂质。PEG脂质可以选自由以下组成的非限制性组：PEG改性的磷脂酰乙醇胺、PEG改性的磷脂酸、PEG改性的神经酰胺(PEG-CER)、PEG改性的二烷基胺、PEG改性的二酰基甘油(PEG-DEG)、PEG改性的二烷基甘油或其组合。例如，聚乙二醇修饰的脂质的实例包括但不限于：1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol，DMG-PEG)、1,2-二油酰基-rac-甘油,甲氧基-聚乙二醇(1,2-Dioleoyl-rac-glycerol,methoxypolyethylene Glycol，DOGPEG)和1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-聚(乙二醇)(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol)，DSPE-PEG)。
在一实施方案中，聚乙二醇修饰的脂质为DMG-PEG，例如DMG-PEG 2000。在一实施方案中，DMG-PEG 2000具有以下结构：
Figure PCTCN2022131289-appb-000021
其中n的平均值为44。
阳离子聚合物
如本文所用，术语“阳离子聚合物”涉及在指定pH下能够带有净正电荷从而与核酸静电结合的任何离子聚合物。阳离子聚合物的实例包括但不限于：聚-L-赖氨酸、鱼精蛋白、聚乙烯亚胺(PEI)或其组合。聚乙烯亚胺可以是线性或支化的聚乙烯亚胺。
术语“鱼精蛋白”是指富含精氨酸的低分子量碱性蛋白，其存在于各种动物(特别是鱼)的精细胞中并代替组蛋白与DNA结合。在一优选实施方案中，阳离子聚合物为鱼精蛋白(例如硫酸鱼精蛋白)。
药物组合物
本发明还提供一种药物组合物，其包含本发明的人工核酸分子、本发明的载体、本发明的细胞或本发明的脂质组合物，以及药学上可接受的载剂。
药学上可接受的载剂可以包括但不限于：稀释剂、粘合剂和胶粘剂、润滑剂、崩解剂、防腐剂、媒介物、分散剂、助流剂、甜味剂、包衣、赋形剂、防腐剂、抗氧化剂(如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠、抗坏血酸棕榈酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚、柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等)、增溶剂、胶凝剂、软化剂、溶剂(例如，水、酒精、乙酸和糖浆)、缓冲剂(例如，磷酸盐缓冲剂、组氨酸缓冲剂和乙酸盐缓冲剂)、表面活性剂(例如非离子表面活性剂，例如聚山梨酯80、 聚山梨酯20、泊洛沙姆或聚乙二醇)、抗细菌剂、抗真菌剂、等渗剂(例如海藻糖、蔗糖、甘露醇、山梨醇、乳糖、葡萄糖)、吸收延迟剂、螯合剂和乳化剂。对于包含人工核酸分子、载体、细胞或脂质组合物的药物组合物而言，合适的载剂可以选自缓冲剂(例如柠檬酸盐缓冲液、乙酸盐缓冲液、磷酸盐缓冲液、组氨酸缓冲液、组氨酸盐缓冲液)、等渗剂(例如海藻糖、蔗糖、甘露醇、山梨醇、乳糖、葡萄糖)、非离子表面活性剂(例如聚山梨酯80、聚山梨酯20、泊洛沙姆)或其组合。
本文提供的药物组合物可以为多种剂型，包括但不限于固体、半固体、液体、粉末或冻干形式。对于包含人工核酸分子、载体、细胞或脂质组合物的药物组合物而言，优选的剂型通常可以为例如注射液和冻干粉。
可通过本领域已知的任何方法，例如通过全身或局部施用，将本文提供的药物组合物给药于受试者。给药途径包括但不限于肠胃外(例如，静脉内、腹膜内、皮内、肌肉内、皮下或腔内)、局部(例如瘤内)、硬膜外或粘膜(例如鼻内、口服、阴道、直肠、舌下或局部)。优选地，所述药物组合物适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮给药(如通过注射或输注)。给药方法可以为例如注射或输注。
治疗
在又一方面，本发明涉及本发明的人工核酸分子、本发明的载体、本发明的细胞、本发明的脂质组合物或本发明的药物组合物在制备疫苗或用于基因治疗的药物中的用途。
在又一方面，本发明涉及本发明的人工核酸分子、本发明的载体、本发明的细胞、本发明的脂质组合物或本发明的药物组合物在制备用于治疗或预防疾病的药物中的用途。
本发明的人工核酸分子、本发明的载体、本发明的细胞、本发明的脂质组合物或本发明的药物组合物可以用于治疗疾病、病症或病况。确切地说，本发明的人工核酸分子、本发明的载体、本发明的细胞、本发明的脂质组合物或本发明的药物组合物可以用于治疗以遗失或异常蛋白或多肽活性为特征的疾病、病症或病况。例如，包含编码遗失或异常多肽的mRNA的人工核酸分子、载体、细胞、脂质组合物或药物组合物可以被施用或递送至细胞。该mRNA随后翻译可以产生所述多肽，由此减少或消除由该多肽的不存在或异常活性引起的问题。由于翻译可以迅速地发生，故这些方法和人工核酸分子、载体、细胞、脂质组合物或药物组合物以用于治疗急性疾病、病症或病况如败血症、中风和心肌梗塞。
可以被施用本发明的人工核酸分子、载体、细胞、脂质组合物或药物组合物的以功能失常或异常蛋白或多肽活性为特征的疾病、病症或病况包括但不限于罕见病、感染性疾病(呈疫苗和治疗剂形式)、癌症和增生性疾病、遗传疾病(例如囊肿性纤维化)、自体免疫疾病、神经退化性疾病、心血管和肾血管疾病、以及代谢性疾病。有多种疾病、病 症或病况可以通过蛋白活性遗失(或大体上降低使得无法出现适当蛋白功能)表征。这些蛋白可能不存在，或者其可能基本上无功能。功能失常的蛋白的具体实例是囊肿性纤维化跨膜传导调控蛋白(CFTR)基因的错义突变变体，这些突变变体产生CFTR蛋白的功能失常的蛋白变体，由此引起囊肿性纤维化。本发明提供了一种通过施用本发明的人工核酸分子、载体、细胞、脂质组合物或药物组合物来治疗受试者的此类疾病、病症或病况的方法，其中RNA可以是编码拮抗或以其它方式克服受试者细胞中存在的异常蛋白活性的多肽的mRNA。
本发明的人工核酸分子、载体、细胞、脂质组合物或药物组合物可以使用任何合理量和任何施用途径施用给受试者，所述合理量和施用途径可有效实现疾病、病症或病况的预防、治疗、诊断或用于任何其它目的。施用至给定受试者的具体量可以取决于受试者的物种、年龄和一般状况；施用目的；具体组合物；施用模式等而变化。
在一些实施方案中，本发明的人工核酸分子、载体、细胞、脂质组合物或药物组合物可以通过本领域技术人员已知的任何方法施用受试者，例如肠胃外、经口、经粘膜、经皮、肌肉内、静脉内、皮内、皮下或腹腔内。
试剂盒
本发明还提供试剂盒，其包含本发明的人工核酸分子、载体、细胞、脂质组合物或药物组合物，以及使用说明。试剂盒还可以包含合适的容器。在某些实施方案中，试剂盒还包含给药的装置。试剂盒一般包括表明试剂盒内容物的预期用途和/或使用方法的标签。术语“标签”包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
有益效果
本发明的人工核酸分子、载体、细胞、脂质组合物或药物组合物可以呈现出优异的效果，例如但不限于：1)提高所包含的mRNA的翻译效率；和/或2)所包含的mRNA具有高稳定性。
实施例
通过参考在此给出的一些具体实施例可获得对本发明的进一步的理解，这些实施例仅用于说明本发明，其无意于对本发明的范围做出任何限制。显然，可以对本发明作出多种改动和变化而不脱离本发明的实质，因此，这些改动和变化同样在本申请要求保护的范围内。本文中所用的比例包括百分比，如果没有特别指出，都按重量计。
实验材料
根据式(I)所述的阳离子脂质为斯微生物合成或者参考，例如CN110520409A、 WO2018081480A1或US11,246,933B1制备；磷脂(DOPE)采购自CordenPharma；胆固醇采购于Sigma-Aldrich；mPEG2000-DMG(即DMG-PEG 2000)采购于Avanti Polar Lipids,Inc.；PBS采购于Invitrogen；硫酸鱼精蛋白采购自北京斯利安药业有限公司；mPEG2000-DSPE采购于lipoid GmbH；DSPC采购于Avanti Polar Lipids,Inc。
实施例1 合成根据式(I)所述的化合物
一般考虑
除非另外指出，否则使用的所有溶剂和试剂都是商购得到并且以原样使用。 1H NMR谱是在300K下使用Bruker Ultrashield 300MHz仪器在CDCl 3中记录。化学位移是关于 1H以相对于TMS(0.00)的百万分率(ppm)报导。硅胶色谱法是在ISCO CombiFlash Rf+Lumen仪器上，使用ISCO RediSep Rf Gold快速柱(粒度：20-40微米)执行。
以下描述的程序可用于合成化合物SW-II-115至SW-II-140-2。
本文采用了以下缩写：
THF:四氢呋喃
MeCN:乙腈
LAH:氢化铝锂
DCM:二氯甲烷
DMAP:4-二甲基氨基吡啶
LDA:二异丙基氨基锂
rt:室温
DME:1,2-二甲氧基乙烷
n-BuLi:正丁基锂
CPME:环戊基甲基醚
EDCI:N-(3-二甲基氨基丙基)-N’-乙基碳酰二亚胺
DIEA:N,N-二异丙基乙胺
PE:石油醚
EA:乙酸乙酯
A.化合物SW-II-115
Figure PCTCN2022131289-appb-000022
1、中间体3的合成
Figure PCTCN2022131289-appb-000023
向含有化合物1(10g,45mmol,1eq.)和化合物2(7.8g,54mmol,1.2eq.)的DCM溶液(100mL)中加入EDCI(17.3g,90mmol,2eq.)和DMAP(2.2g,18mmol,0.4eq.)，然后加入DIEA(23.2g,180mmol,4eq.)。将反应混合物在室温下在N 2保护下搅拌16小时。TLC(石油醚:乙酸乙酯＝30:1)显示化合物1被消耗并且形成了所需产物。反应混合物用DCM(20mL)稀释并用H 2O(40mL)洗涤，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚:乙酸乙酯(1:0-20:1)洗脱，得到无色油状化合物3(4.365g，28％)。
2、中间体5的合成
Figure PCTCN2022131289-appb-000024
将化合物3(500mg,1.437mmol,1eq.)和化合物4(2.63g,43.103mmol,30eq.)的EtOH溶液在N 2保护下在60℃下搅拌16小时。TLC(DCM:MeOH＝10:1)显示化合物3被消耗，TLC(DCM/MeOH＝10/1)显示观察到新的主要点。在减压下浓缩反应混合物。残余物用EtOAc(50mL)稀释并用H 2O(3×50mL)洗涤。有机层用无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1:0-10:1,v/v)洗脱，得到黄色油状化合物5(264mg，56％)。
3、中间体8的合成
Figure PCTCN2022131289-appb-000025
向化合物6(500mg,1.712mmol,1eq.)和化合物7(1.113g,8.562mmol,5eq)在二氧六环/水(5mL/0.5mL)混合溶剂中加入Pd(dppf)Cl 2(112mg,0.171mmol,0.1eq.)和碳酸钾(709mg,5.136mmol,3eq.)。将混合物在N 2下于100℃搅拌过夜。TLC(PE:EA＝15:1)显示反应完成并观察到新的主要点。混合物用EA萃取并用水洗涤，有机层用无水Na 2SO 4干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE:EA(1:0-10:1)洗脱，得到无色油状化合物8(455mg，88％)。
4、中间体9的合成
Figure PCTCN2022131289-appb-000026
在0℃和N 2保护下，向化合物8(455mg,1.497mmol,1eq.)的THF(5mL)溶液中加入LiAlH 4(1.5mL,1.497mmol,1M，THF中,1eq.)。将混合物在室温下在N 2下搅拌2小时。TLC(PE:EtOAc＝5:1)显示反应完成并观察到新的主要点。混合物用水(1.5mL)淬灭并用2N HCl处理以将PH调节在6和7之间，用EA萃取并用盐水洗涤。有机层用无水Na 2SO 4干燥，过滤并真空浓缩，得到粗品化合物9(419mg，>100％)，为无色油状物，无需进一步纯化。
5、中间体10的合成
Figure PCTCN2022131289-appb-000027
向含有化合物1(339mg,1.518mmol,1eq.)和化合物9(419mg,1.518mmol,1eq.)的DCM(4mL)溶液中加入EDCI(583mg,3.036mmol,2eq.)和DMAP(74mg,0.607mmol,0.4eq.)，然后加入DIEA(783mg,6.072mmol,4eq.)。将反应混合物在室温下在N 2保护下搅拌16小时。TLC(石油醚:乙酸乙酯＝10:1)显示形成了所需产物。反应混合物用EA萃取并用水洗涤。有机层用无水Na 2SO 4干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚:乙酸乙酯(1:0-10:1)洗脱，得到化合物10(443mg，60.7％)，为无色油。
6、终产物SW-II-115的合成
Figure PCTCN2022131289-appb-000028
向含有化合物10(307mg,0.64mmol,1eq.)和化合物5(210mg,0.64mmol,1eq.)的混合溶剂CPME/CH 3CN(3mL/3mL)中加入K 2CO 3(530mg,3.84mmol,6eq.)和KI(212mg,1.28mmol,2eq.)。添加完毕后，将混合物在N 2下在90℃下搅拌过夜。TLC(DCM:MeOH＝10:1)显示反应完成并观察到新的主要点。混合物用EA萃取并用水洗涤。有机层用无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM:MeOH(1:0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-115(266mg,57％)。
LCMS:Rt:1.293min；MS m/z(ELSD):730.5[M+H] +；
HPLC:99.472％纯度，ELSD；RT＝4.895min.
1H NMR(400MHz,CDCl 3)δ7.21–6.99(m,3H),5.05(s,2H),4.05(t,J＝6.8Hz,2H),3.58(t,J＝5.3Hz,2H),2.69–2.46(m,10H),2.31(dt,J＝20.0,7.5Hz,4H),1.69–1.18(m, 51H),0.89(dt,J＝12.4,6.3Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.90(s),173.68(s),140.80(d,J＝13.0Hz),133.31(s),129.25(d,J＝16.2Hz),128.30(s),125.75(s),77.30(d,J＝11.5Hz),77.04(s),76.72(s),66.22(s),64.43(s),58.12(s),55.72(s),53.90(s),34.32(d,J＝1.9Hz),32.69(s),32.48(s),31.81(d,J＝11.2Hz),31.25(s),29.59–28.91(m),28.66(s),27.17(s),26.64(s),25.94(s),24.91(d,J＝5.1Hz),22.65(d,J＝3.3Hz),14.10(s).
B.化合物SW-II-118
Figure PCTCN2022131289-appb-000029
1、中间体3的合成
Figure PCTCN2022131289-appb-000030
化合物1(1.22g,5.0mmol,1.0eq.)和化合物2(765mg,7.5mmol,1.5eq.)、Pd(PPh 3) 4(四三苯基膦钯，289mg,0.25mmol,0.05eq.)和K 2CO 3(1.38g,10.0mmol,2.0eq.)的甲苯(10ml)和H 2O(1ml)溶液在110℃下，N 2保护下搅拌1小时。TLC(石油醚:乙酸乙酯＝19:1)显示化合物1被消耗并且观察到一个新点。反应混合物用DCM(50mL)稀释并用H 2O(40mL)洗涤，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚:乙酸乙酯(1:0-10:1)洗脱，得到无色油状化合物3(0.5g，45％)。
1H NMR(400MHz,CDCl 3)δ7.16(dd,J＝23.5,8.1Hz,4H),4.14(q,J＝7.1Hz,2H),3.57(s,2H),2.64–2.48(m,2H),1.66–1.51(m,2H),1.35(dd,J＝15.0,7.4Hz,2H),1.25(t,J＝7.1Hz,3H),0.92(t,J＝7.3Hz,3H).
2、中间体4的合成
Figure PCTCN2022131289-appb-000031
在-78℃下将LiAlH 4(193mg，5.09mmol，4.0eq.)加入到含有化合物3(280mg，1.27mmol，1.0eq.)THF(10mL)溶液，然后将反应在10℃下反应3小时。TLC显示反应很好，将反应浓缩并用Na 2SO 4(20mL)稀释并用EA(30mLx2)萃取，有机相用无水Na 2SO 4干燥，过滤并减压浓缩，得到黄色油状化合物4(3.12g，粗品)。
3、中间体6的合成
Figure PCTCN2022131289-appb-000032
含有化合物4(215mg,1.2mmol,1.0eq.)、化合物5(404mg,1.8mmol,1.5eq.)、EDCI(1.15g,6.0mmol,5.0eq.)、DMAP(732mg,1.8eq.)，DIEA(1.29g,12.0mmol,10.0eq.)和DIEA(1.29g,12.0mmol,10.0eq.)的DCM(5mL)溶液在N 2保护下，10℃下搅拌16h。TLC(DCM:MeOH＝10:1)显示反应完成并观察到新的主要点。减压浓缩混合物，残余物通过硅胶柱色谱纯化，用PE:EA(1:0-10:1,v/v)洗脱，得到无色油状化合物6(145mg,31％)。
1H NMR(400MHz,CDCl 3)δ7.12(s,4H),4.27(t,J＝7.1Hz,2H),3.52(t,J＝6.7Hz,1H),3.40(t,J＝6.8Hz,1H),2.90(t,J＝7.1Hz,2H),2.65–2.50(m,2H),2.28(t,J＝7.5Hz,2H),1.93–1.70(m,2H),1.64–1.56(m,4H),1.44–1.27(m,8H),0.92(t,J＝7.3Hz,3H).
4、终产物SW-II-118的合成
Figure PCTCN2022131289-appb-000033
含有化合物6(140mg,0.37mmol,1.0eq.)、化合物7(243mg,0.55mmol,1.5eq.)、K 2CO 3(153mg,1.11mmol,3.0eq.)和KI(123mg,0.74mmol,2.0eq.)的混合物在CPME(1mL)和CH 3CN(1mL)混合溶剂在N 2下在90℃搅拌16小时。减压浓缩反应混合物，残余物用EtOAc(50mL)稀释并用NaHCO 3(30mL)洗涤。有机层用无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM:MeOH(1:0-10:1，v/v)洗脱，得到呈黄色油状SW-II-118(105mg,61％)。
LCMS:Rt:1.946min；MS m/z(ELSD):744.4[M+H] +；
HPLC:99.64％纯度，ELSD；RT＝5.875min.
1H NMR(400MHz,CDCl 3)δ7.11(s,4H),4.91–4.79(m,1H),4.26(t,J＝7.2Hz,2H),3.80–3.68(m,2H),2.90(t,J＝7.1Hz,4H),2.81–2.67(m,4H),2.62–2.52(m,2H),2.28(td,J＝7.5,2.6Hz,4H),1.64–1.51(m,11H),1.38–1.17(m,42H),0.93–0.82(m,9H).
13C NMR(101MHz,CDCl 3)δ173.61(d,J＝11.7Hz),141.11(s),134.90(s),128.74(s),128.51(s),77.40(s),77.08(s),76.77(s),74.17(s),64.90(s),57.48(s),56.24(s),53.98(s),35.25(s),34.66(d,J＝14.4Hz),34.16(d,J＝5.1Hz),33.67(s),31.86(s),29.52(d,J＝2.4Hz),29.24(s),29.21–28.74(m),26.90(d,J＝4.9Hz),25.42–24.92(m),24.92–24.88(m),24.74(s),22.67(s),22.37(s),14.04(d,J＝15.7Hz).
C.化合物SW-II-120
Figure PCTCN2022131289-appb-000034
1、中间体3的合成
Figure PCTCN2022131289-appb-000035
含有化合物1(1.22g,5.0mmol,1.0eq.)、化合物2(1.30mg,10.0mmol,2.0eq.)、Pd(PPh 3) 4(289mg,0.25mmol,0.05eq.)和K 2CO 3(1.38g,10.0mmol,2.0eq.)在甲苯(10ml)和H 2O(1ml)的混合溶液中在110℃下，N 2保护下搅拌1小时。TLC(石油醚:乙酸乙酯＝19:1)显示化合物1被消耗并且观察到一个新点。反应混合物用DCM(50mL)稀释并用H 2O(40mL)洗涤，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚:乙酸乙酯(1:0-10:1)洗脱，得到无色油状化合物3(0.78g，62％)。
1H NMR(400MHz,CDCl 3)δ7.19(d,J＝8.1Hz,2H),7.13(d,J＝8.1Hz,2H),4.14(q,J＝7.1Hz,2H),3.57(s,2H),2.62–2.51(m,2H),1.58(d,J＝11.1Hz,2H),1.35–1.21(m,9H),0.88(t,J＝6.7Hz,3H).
2、中间体4的合成
Figure PCTCN2022131289-appb-000036
在-78℃下将LiAlH 4(477mg,12.56mmol,4.0eq.)加入到含有化合物3(780mg,3.14mmol,1.0eq.)的THF(10mL)溶液中，然后将反应在10℃下搅拌3小时。薄层色谱显示反应进行得很好。将反应浓缩并用Na 2SO 4(20mL)稀释并用EA(30mL*2)萃取，有机相用无水Na 2SO 4干燥，过滤并减压浓缩，得到无色油状化合物4(640mg，粗品)。
3、中间体6的合成
Figure PCTCN2022131289-appb-000037
含有化合物4(640mg,3.10mmol,1.0eq.)、化合物5(1.06g,4.70mmol,1.5eq.)、EDCI(2.98g,15.5mmol,5.0eq.)、DMAP(1.85g,15.0eq.)和DIEA(4.0g,31.0mmol,10.0eq.)的 DCM(10mL)溶液，在N 2保护下，在10℃下搅拌16h。TLC(DCM:MeOH＝10:1)显示反应完成并观察到新的主要点。减压浓缩混合物，残余物通过硅胶柱色谱纯化，用PE:EA(1:0-10:1，v/v)洗脱，得到无色油状化合物6(465mg，36％)。
4、终产物SW-II-120的合成
Figure PCTCN2022131289-appb-000038
含有化合物6(100mg,0.25mmol,1.0eq.)、化合物7(161mg,0.36mmol,1.5eq.)、K 2CO 3(104mg,0.75mmol,3.0eq.)和KI(83mg,0.50mmol,2.0eq.)的混合物在CPME(1mL)和CH 3CN(1mL)在N 2下在90℃搅拌16小时。在减压下浓缩反应混合物，残余物用EtOAc(50mL)稀释并用NaHCO 3(30mL)洗涤。有机层用无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM:MeOH(1:0-10:1，v/v)洗脱，得到呈黄色油状的SW-II-120(100mg，52％)。
LCMS:Rt:2.500min；MS m/z(ELSD):772.4[M+H] +；
HPLC:99.70％纯度，ELSD；RT＝8.675min.
1H NMR(400MHz,CDCl 3)δ7.07(d,J＝8.9Hz,4H),4.89–4.73(m,1H),4.23(t,J＝7.2Hz,2H),3.83–3.65(m,2H),2.87(t,J＝7.2Hz,4H),2.82–2.67(m,4H),2.61–2.45(m,2H),2.25(td,J＝7.5,2.5Hz,4H),1.65–1.44(m,15H),1.27(dd,J＝13.2,11.3Hz,42H),0.85(t,J＝6.8Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.57(d,J＝11.5Hz),141.13(s),134.88(s),128.73(s),128.48(s),77.45(s),77.13(s),76.81(s),74.14(s),64.89(s),57.34(s),56.17(s),53.92(s),35.57(s),34.64(d,J＝16.1Hz),34.14(d,J＝3.3Hz),31.79(d,J＝13.4Hz),31.49(s),29.50(d,J＝2.2Hz),29.23(s),29.10–28.71(m),26.85(d,J＝5.0Hz),25.49–25.38(m),25.13(d,J＝35.4Hz),24.72(s),22.63(d,J＝5.8Hz),14.11(s).
D.化合物SW-II-121
Figure PCTCN2022131289-appb-000039
1、中间体3的合成
Figure PCTCN2022131289-appb-000040
向含有化合物1(1.3g,5.86mmol,1.5eq.)和化合物2(1g,3.9mmol,1.0eq.)的DCM (20mL)溶液中加入EDCI(1.495g,7.8mmol,2.0eq.)、DMAP(0.19g,1.56mmol,0.4eq.)和DIEA(2.57mL,15.6mmol,4.0eq.)。将反应混合物在室温下在N 2下搅拌16小时。TLC(石油醚:乙酸乙酯＝19:1)显示化合物2被消耗并且形成了所需产物。反应混合物用DCM(20mL)稀释并用H 2O(40mL)洗涤，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚:乙酸乙酯(1:0-10:1)洗脱，得到黄色油状化合物3(1.2g，66.9％)。
1H NMR(400MHz,CDCl 3)δ4.92–4.82(m,1H),3.42(t,J＝6.8Hz,2H),2.31(t,J＝7.5Hz,2H),1.95–1.82(m,2H),1.70–1.19(m,36H),0.90(t,J＝6.8Hz,6H).
2、中间体5的合成
Figure PCTCN2022131289-appb-000041
含有化合物3(5.2g,11.30mmol,1.0eq.)和化合物4(20.6g,339mmol,30eq.)的EtOH(5mL)溶液在N 2保护下于60℃搅拌16小时。TLC(石油醚:乙酸乙酯＝19:1)显示化合物3被消耗并且TLC(DCM/MeOH＝10/1)显示观察到新的主要点。在减压下浓缩反应混合物，残余物用EtOAc(50mL)稀释并用H 2O(3×50mL)洗涤。有机层用无水Na 2SO 4干燥，过滤并减压浓缩，残余物通过硅胶柱色谱纯化，用DCM:MeOH(1:0-10:1，v/v)洗脱，得到黄色油状化合物5(3g，60％)。
3、中间体8的合成
Figure PCTCN2022131289-appb-000042
含有化合物6(1g,4.115mmol,1eq.)和化合物7(889mg,6.173mmol,1.5eq)的甲苯/水(10mL/1mL)混合溶液中加入Pd(pph 3) 4(238mg,0.206mmol,0.05eq.)、K 2CO 3(1.7g,12.35mmol,3eq)。将混合物在N 2和110℃下搅拌2小时。TLC(PE:EA＝10:1)显示反应完成并观察到新的主要点。混合物用EA萃取并用水洗涤。有机层用无水Na 2SO 4干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE:EA(1:0-10:1)洗脱，得到无色油状化合物8(714mg,66％)。
4、中间体9的合成
Figure PCTCN2022131289-appb-000043
N 2保护下，在0℃下向化合物8(714mg,2.725mmol,1eq.)的THF(7mL)溶液中的混合物中加入LiAlH 4(2.7mL,2.725mmol,1M,THF中，1eq.)，混合物在室温下搅拌2小时。TLC(PE:EtOAc＝10:1)显示反应完成并观察到新的主要点。混合物用水(2.7mL)淬灭并用2N HCl处理以将PH调节在6和7之间，用EA萃取并用盐水洗涤。有机层用无水 Na 2SO 4干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE:EA(1:0-10:1)洗脱，得到无色油状化合物9(103mg，63％)。
5、中间体11的合成
Figure PCTCN2022131289-appb-000044
向含有化合物9(300mg,1.364mmol,1eq.)和化合物10(363mg,1.64mmol,1.2eq.)的DCM(3mL)中加入EDCI(524mg,2.728mmol,2eq.)、DMAP(67mg,0.546mmol,0.4eq.)，和DIEA(704mg,5.456mmol,4eq.)。将反应混合物在室温下在N 2下搅拌16小时。TLC(石油醚:乙酸乙酯＝10:1)显示形成了所需产物。反应混合物用EA萃取并用水洗涤。有机层用无水Na 2SO 4干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚:乙酸乙酯(1:0-10:1)洗脱，得到无色油状化合物11(169mg，29％)。
6、终产物SW-II-121的合成
Figure PCTCN2022131289-appb-000045
向含有化合物11(169mg,0.399mmol,1eq.)和化合物5(176mg,0.399mmol,1eq.)的CPME/CH 3CN(2mL/2mL)混合溶剂中加入K 2CO 3(330mg,2.394mmol,6eq.)和KI(132mg,0.798mmol,2eq.)。添加完毕后，将混合物在N 2下在90℃下搅拌过夜。TLC(DCM:MeOH＝10:1)显示反应完成并观察到新的主要点。混合物用EA萃取并用水洗涤，有机层用无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM:MeOH(1:0-10:1，v/v)洗脱，得到黄色油状化合物SW-II-121(145mg，46％)。
LCMS:Rt:1.493min；MS m/z(ELSD):786.5[M+H] +；
HPLC:99.869％纯度，ELSD；RT＝10.655min.
1H NMR(400MHz,CDCl 3)δ7.11(s,4H),4.92–4.80(m,1H),4.26(t,J＝7.2Hz,2H),3.80(s,2H),2.87(dd,J＝26.6,19.4Hz,7H),2.62–2.51(m,2H),2.28(td,J＝7.2,3.6Hz,4H),1.75–1.45(m,14H),1.42–1.09(m,45H),0.88(t,J＝6.8Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.61(d,J＝12.3Hz),141.20(s),134.90(s),128.75(s),128.51(s),77.35(s),77.03(s),76.72(s),74.21(s),64.93(s),54.15(s),35.59(s),34.66(d,J＝16.6Hz),34.16(d,J＝3.0Hz),31.85(d,J＝4.4Hz),31.55(s),29.64–29.15(m),29.15–28.78(m),26.85(d,J＝4.5Hz),25.33(s),24.95(s),24.72(s),22.68(s),14.12(s).
E.化合物SW-II-122
Figure PCTCN2022131289-appb-000046
1、化合物3的合成
Figure PCTCN2022131289-appb-000047
化合物1(1g,4.65mmol,1eq.)和化合物2(726mg,5.58mmol,1.2eq.)溶解在甲苯/水(10/1,20mL)中，然后向该混合物中加入K 2CO 3(1.92g,13.9mmol,3eq.)和Pd(pph 3) 4(269mg,0.23mmol,0.05eq)。将反应混合物置于N 2中加热至110℃搅拌2小时。TLC(石油醚/乙酸乙酯＝19/1)显示化合物1被消耗并且观察到新的主要斑点。反应混合物用H 2O(80mL)淬灭并且用乙酸乙酯(60mL×3)萃取，有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-10/1)洗脱，得到黄色油状化合物3(800mg,78％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000048
在氮气保护0℃下，向溶解在THF(14mL)的化合物3(700mg,3.18mmol,1.0eq.)中加入LiAlH 4(3.2mL,3.18mmol,1eq)。反应物升至室温氮气保护下搅拌2小时。TLC(PE/EtOAc＝10/1)显示反应完成并观察到新的主要斑点。混合物分别用水(3.2mL)和1M HCl(3.2mL)淬灭。再向混合物中加入水(6mL)，乙酸乙酯(60mL×3)萃取。有机层用盐水(30mL×2)洗涤，无水硫酸钠干燥，过滤，并减压浓缩。残余物通过硅胶柱色谱纯化，用乙酸乙酯/石油醚＝1/10洗脱，得到黄色油状化合物4(600mg,98％)。
3、化合物6的合成
Figure PCTCN2022131289-appb-000049
化合物4(680mg,3.5mmol,1.0eq.)和化合物5(1.13g,5.1mmol,1.5eq.)溶解在DCM(10mL)中，向该混合物中加入EDCI(1.20g,6.25mmol,2.0eq.)，DMAP(166mg,1.36mmol,0.4eq.)和DIEA(1.78g,13.8mmol,4.0eq.)。添加完后，反应混合物在氮气保护下室温搅拌过夜。TLC(DCM/MeOH＝30/1)显示起始材料被消耗并且形成了一个新斑点。混合物用水(70mL)淬灭并用DCM(80mL×3)萃取。合并的有机层用盐水(2×20mL)洗涤，用无 水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用乙酸乙酯/石油醚＝3/97溶液洗脱，得到黄色油状化合物6(680mg,48.5％)。
4、SW-II-122的合成
Figure PCTCN2022131289-appb-000050
化合物6(108mg,0.27mmol,1.2eq)和化合物7(100mg,0.23mmol,1eq.)溶解在CPME(2mL)和CH 3CN(2mL)中，向该混合物中加入碳酸钾(157mg,1.14mmol,5.0eq)和碘化钾(75mg,0.45mmol,2.0eq)。添加完后，反应混合物在氮气保护下90℃搅拌16小时。TLC(DCM/MeOH＝10/1)显示反应完成。减压浓缩反应混合物。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到SW-II-122(68mg,40％)，为无色油状物。
LCMS:Rt:1.487min；MS m/z(ELSD):758.5[M+H] +；
HPLC:97.3％纯度，ELSD；RT＝7.622min.
1H NMR(400MHz,CDCl 3)δ7.32(d,J＝26.4Hz,1H),7.17(dd,J＝27.2,21.1Hz,3H),5.09(s,2H),4.91–4.79(m,1H),3.85(s,2H),2.98(s,2H),2.87(s,4H),2.65–2.54(m,2H),2.35(t,J＝7.6Hz,2H),2.28(t,J＝7.6Hz,2H),1.74–1.57(m,9H),1.50(d,J＝5.6Hz,4H),1.37–1.15(m,43H),0.94–0.80(m,9H).
13C NMR(101MHz,CDCl 3)δ173.55(d,J＝2.4Hz),143.35(s),135.92(s),128.67–128.19(m),125.47(s),77.36(s),77.04(s),76.73(s),74.22(s),66.27(s),57.15(s),56.74(s),54.14(s),35.88(s),34.55(s),34.15(d,J＝3.6Hz),31.79(d,J＝15.2Hz),31.43(s),29.52(d,J＝2.8Hz),29.25(s),28.92(dd,J＝14.2,5.8Hz),26.77(d,J＝4.8Hz),25.33(s),24.92(s),24.71(s),24.48(s),22.64(d,J＝6.8Hz),14.12(s).
F.化合物SW-II-127
Figure PCTCN2022131289-appb-000051
1、化合物3的合成
Figure PCTCN2022131289-appb-000052
化合物1(1.3g,5.86mmol,1.5eq.)和化合物2(1g,3.9mmol,1.0eq.)溶解在DCM(20mL)中，向该混合物中加入EDCI(1.495g,7.8mmol,2.0eq.)和DMAP(0.19g,1.56mmol,0.4eq.)，然后加入DIEA(2.57mL,15.6mmol,4.0eq.)。反应混合物在氮气保护下室温搅拌16 小时。TLC(石油醚/乙酸乙酯＝19/1)显示化合物2被消耗并且形成了所需产物。反应混合物用DCM(20mL)稀释并用H 2O(40mL)洗涤，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-10/1)洗脱，得到黄色油状化合物3(1.2g，66.9％)。
1H NMR(400MHz,CDCl 3)δ4.92–4.82(m,1H),3.42(t,J＝6.8Hz,2H),2.31(t,J＝7.5Hz,2H),1.95–1.82(m,2H),1.70–1.19(m,36H),0.90(t,J＝6.8Hz,6H).
2、化合物5的合成
Figure PCTCN2022131289-appb-000053
将化合物3(5.2g,11.30mmol,1.0eq.)和化合物4(20.6g,339mmol,30eq.)加入到EtOH(5mL)中，然后混合物在氮气保护下60℃搅拌16小时。TLC(石油醚/乙酸乙酯＝19/1)显示化合物3被消耗并且TLC(DCM/MeOH＝10/1)显示观察到新的主要斑点。减压下浓缩反应混合物。残余物用EtOAc(50mL)稀释并用H 2O(3X50mL)洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1，v/v)洗脱，得到黄色油状的化合物5(3g，60％)。
1H NMR(400MHz,CDCl 3)δ4.95–4.75(m,1H),3.74–3.58(m,2H),2.87–2.74(m,2H),2.69–2.56(m,2H),2.36(s,2H),2.28(t,J＝7.5Hz,2H),1.65–1.42(m,8H),1.38–1.17(m,30H),0.88(t,J＝6.8Hz,6H).
3、化合物8的合成
Figure PCTCN2022131289-appb-000054
化合物7(522mg,2.5mmol,1.2eq.)和化合物6(400mg,2.083mmol,1eq.)溶解在DCM(4mL)中，向该混合物中加入EDCI(800mg,4.166mmol,2eq.)，DMAP(102mg,0.833mmol,0.4eq.)和DIEA(1.075mg,8.332mmol,4eq.)。添加完后，反应混合物在氮气保护下室温搅拌过夜。TLC(PE:EA＝10:1)显示起始材料被消耗并且形成了一个新斑点。减压浓缩反应混合物。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-10/1)洗脱，得到无色油状的化合物8(454mg,57％)。
4、SW-II-127的合成
Figure PCTCN2022131289-appb-000055
化合物8(100mg,0.262mmol,1eq.)和化合物5(139mg,0.314mmol,1.2eq.)溶解在CPME/CH3CN(1mL/1mL)中，向该混合物中加入碳酸钾(217mg,1.572mmol,6eq.)和碘化钾(87mg,0.524mmol,2eq.)。添加完后，反应混合物在氮气保护下90℃搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完全并且形成了所需产物。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-127(42.49mg,22％)。
LCMS:Rt:1.323min；MS m/z(ELSD):744.5[M+H] +；
HPLC:99.742％纯度，ELSD；RT＝7.339min.
1H NMR(400MHz,CDCl 3)δ7.25(s,2H),7.17(d,J＝8.0Hz,2H),5.07(s,2H),4.91–4.82(m,1H),3.83(s,2H),2.90(d,J＝44.8Hz,5H),2.64–2.55(m,2H),2.35(t,J＝7.4Hz,2H),2.28(t,J＝7.5Hz,2H),1.76–1.46(m,14H),1.42–1.19(m,41H),0.88(t,J＝6.8Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.50(d,J＝8.5Hz),133.17(s),128.61(s),128.34(s),77.29(d,J＝11.4Hz),77.03(s),76.71(s),74.23(s),66.19(s),54.20(s),35.71(s),34.56(s),34.10(d,J＝8.8Hz),31.80(d,J＝15.4Hz),31.43(s),29.53(d,J＝2.5Hz),29.25(s),28.95(d,J＝10.5Hz),28.63(s),26.71(d,J＝18.2Hz),25.33(s),24.93(s),24.62(s),22.65(d,J＝6.6Hz),14.13(s).
G.化合物SW-II-134-1
Figure PCTCN2022131289-appb-000056
1、化合物3的合成
Figure PCTCN2022131289-appb-000057
向化合物1(500mg,2.283mmol,1eq.)和化合物2(890mg,6.849mmol,3eq)在甲苯/水(5mL/1mL)中的混合物中加入醋酸钯(51mg,0.228mmol,0.1eq.)、Ruphos(213mg,0.457mmol,0.2eq.)和碳酸钾(945mg,6.849mmol,3eq)。将混合物在氮气下于110℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物3(723mg，99.6％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000058
在0℃和氮气环境下，向化合物3(723mg,2.27mmol,1eq.)在THF(8mL)中的混合物中加入氢化铝锂(2.3mL,2.27mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(2.3mL)淬灭并用2N盐酸处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物4(381mg，58％)，无需进一步纯化。
3、化合物6的合成
Figure PCTCN2022131289-appb-000059
向化合物4(381mg,1.3mmol,1eq.)和化合物5(352mg,1.6mmol,1.2eq.)在DCM(4mL)中的混合物中加入EDCI(499mg,2.6mmol,2eq.)和DMAP(63mg,0.52mmol,0.4eq.)，然后加入DIEA(671mg,5.2mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝20/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到无色油状化合物6(272mg，44％)。
4、SW-II-134-1的合成
Figure PCTCN2022131289-appb-000060
向化合物6(150mg,0.303mmol,1eq.)和化合物7(110mg,0.333mmol,1.1eq.)在CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(251mg,1.818mmol,6eq.)和碘化钾(101mg,0.61mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝15/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1，v/v)洗脱，得到黄色油状化合物SW-II-134-1(168mg，75％)。
LCMS:Rt:1.276min；MS m/z(ELSD):744.4[M+H] +；
HPLC:98.481％纯度，ELSD；RT＝10.724min.
1H NMR(400MHz,CDCl 3)δ7.06(d,J＝7.6Hz,1H),7.01–6.93(m,2H),4.25(t,J＝7.3Hz,2H),4.05(t,J＝6.8Hz,2H),3.85–3.72(m,2H),2.98–2.69(m,8H),2.62–2.48(m,4H),2.29(t,J＝7.5Hz,4H),1.72–1.48(m,14H),1.45–1.17(m,36H),0.89(dt,J＝11.9,6.0Hz,9H).
13CNMR(101MHz,CDCl 3)δ173.78(d,J＝16.7Hz),140.72(s),138.81(s),134.91(s), 129.70(s),129.22(s),126.19(s),77.30(d,J＝11.4Hz),77.03(s),76.72(s),65.02(s),64.49(s),57.42(s),56.36(s),54.08(s),34.76(s),34.22(d,J＝4.2Hz),32.74(s),32.36(s),31.81(d,J＝9.1Hz),31.35(d,J＝5.3Hz),29.49(d,J＝2.8Hz),29.24(d,J＝2.2Hz),28.92(s),28.66(s),26.86(s),25.93(s),25.04(s),24.78(d,J＝6.6Hz),22.65(d,J＝2.6Hz),14.10(s).
H.化合物SW-II-134-2
Figure PCTCN2022131289-appb-000061
1、化合物3的合成
Figure PCTCN2022131289-appb-000062
向化合物1(500mg,2.283mmol,1eq.)和化合物2(1.08g,6.849mmol,3eq)在甲苯/水(5mL/1mL)中的混合物中加入醋酸钯(51mg,0.228mmol,0.1eq.)、Ruphos(213mg,0.457mmol,0.2eq.)和碳酸钾(945mg,6.849mmol,3eq)。将混合物在氮气下于110℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物3(854mg,100％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000063
在0℃和氮气环境下，向化合物3(854mg,2.28mmol,1eq.)在THF(9mL)中的混合物中加入氢化铝锂(2.3mL,2.28mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(2.3mL)淬灭并用2N盐酸处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物4(724mg,92％)，无需进一步纯化。
3、化合物6的合成
Figure PCTCN2022131289-appb-000064
向化合物4(724mg,2.09mmol,1eq.)和化合物5(560mg,2.51mmol,1.2eq.)在DCM(8mL)中的混合物中加入EDCI(803mg,4.18mmol,2eq.)和DMAP(102mg,0.84mmol,0.4eq.)，然后加入DIEA(1.078g,8.36mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝20/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到无色油状化合物6(473mg,41％)。
4、SW-II-134-2的合成
Figure PCTCN2022131289-appb-000065
向化合物6(150mg,0.27mmol,1eq.)和化合物7(108mg,0.33mmol,1.1eq.)在CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(225mg,1.63mmol,6eq.)和碘化钾(90mg,0.54mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝15/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1，v/v)洗脱，得到黄色油状化合物SW-II-134-2(71.77mg,33％)。
LCMS:Rt:1.527min；MS m/z(ELSD):800.4[M+H] +；
HPLC:97.311％纯度，ELSD；RT＝9.025min.
1H NMR(400MHz,CDCl 3)δ7.06(d,J＝7.6Hz,1H),6.96(d,J＝9.6Hz,2H),4.25(t,J＝7.3Hz,2H),4.05(t,J＝6.8Hz,2H),3.80–3.66(m,2H),2.86(dd,J＝12.8,5.6Hz,4H),2.78–2.67(m,4H),2.60–2.52(m,4H),2.29(t,J＝7.5Hz,4H),1.57(dt,J＝15.8,7.3Hz,14H),1.30(d,J＝20.3Hz,45H),0.88(t,J＝6.7Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.82(d,J＝16.9Hz),140.73(s),138.82(s),134.91(s),129.71(s),129.23(s),126.19(s),77.36(s),77.14(d,J＝20.4Hz),76.72(s),65.03(s),64.49(s),57.57(s),56.13(s),54.02(s),34.76(s),34.25(d,J＝4.2Hz),32.76(s),32.37(s),31.89(d,J＝5.3Hz),31.40(d,J＝6.0Hz),29.84(d,J＝3.7Hz),29.63–29.14(m),28.97(s),28.65(s),26.93(s),25.66(d,J＝54.4Hz),24.80(d,J＝6.6Hz),22.68(d,J＝1.8Hz),14.12(s).
I.化合物SW-II-134-3
Figure PCTCN2022131289-appb-000066
1、化合物3的合成
Figure PCTCN2022131289-appb-000067
向化合物1(10g,45mmol,1eq.)和化合物2(7.8g,54mmol,1.2eq.)在DCM(100mL)中的混合物中加入EDCI(17.3g,90mmol,2eq.)和DMAP(2.2g,18mmol,0.4eq.)，然后加入DIEA(23.2g,180mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝30/1)显示化合物1被消耗并且形成了所需产物。反应混合物用乙酸乙酯(20mL)萃取并用水(40mL×3)洗涤，经无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到无色油状化合物3(4.365g,28％)。
2、化合物5的合成
Figure PCTCN2022131289-appb-000068
将化合物3(5g,14.38mmol,1eq.)和化合物4(8.8g,143.7mmol,10eq.)在乙醇(2mL)中的混合物在55℃下在氮气下搅拌16小时。TLC(DCM/MeOH＝10/1)显示观察到新的主要斑点。反应混合物用乙酸乙酯(50mL)萃取并用水(3×50mL)洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1，v/v)洗脱，得到黄色油状化合物5(1.008g，21％)。
3、化合物8的合成
Figure PCTCN2022131289-appb-000069
向化合物6(500mg,2.283mmol,1eq.)和化合物7(699mg,6.849mmol,3eq)在甲苯/水(5mL/1mL)中的混合物中加入醋酸钯(51mg,0.228mmol,0.1eq.)、Ruphos(213mg,0.457mmol,0.2eq.)和碳酸钾(945mg,6.849mmol,3eq)。将混合物在氮气下于110℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物8(507mg,85％)。
4、化合物9的合成
Figure PCTCN2022131289-appb-000070
在0℃和氮气环境下，向化合物8(507mg,1.935mmol,1eq.)在THF(5mL)中的混合物中加入氢化铝锂(2mL,1.935mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(2mL)淬灭并用2N盐酸处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物9(492mg,>100％)，无需进一步纯化。
5、化合物10的合成
Figure PCTCN2022131289-appb-000071
向化合物9(492mg,2.103mmol,1eq.)和化合物1(563mg,2.523mmol,1.2eq.)在DCM(5mL)中的混合物中加入EDCI(808mg,4.206mmol,2eq.)和DMAP(103mg,0.84mmol,0.4eq.)，然后加入DIEA(1.085g,8.412mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝15/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-10/1)洗脱，得到无色油状化合物10(329mg,36％)。
6、SW-II-134-3的合成
Figure PCTCN2022131289-appb-000072
向化合物10(150mg,0.34mmol,1eq.)和化合物5(134mg,0.41mmol,1.2eq.)在CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(282mg,2.04mmol,6eq.)和碘化钾(113mg,0.68mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-134-3(63.59mg,25％)。
LCMS:Rt:1.247min；MS m/z(ELSD):688.3[M+H] +；
HPLC:95.945％纯度，ELSD；RT＝6.186min.
1H NMR(400MHz,CDCl 3)δ7.07(d,J＝7.6Hz,1H),6.97(dd,J＝9.9,2.2Hz,2H),4.26(t,J＝7.2Hz,2H),4.05(t,J＝6.8Hz,2H),2.88(dd,J＝14.8,7.6Hz,4H),2.78–2.74(m,2H),2.67–2.54(m,8H),2.29(t,J＝7.5Hz,4H),1.68–1.47(m,15H),1.37–1.22(m,27H),0.98–0.86(m,9H).
13C NMR(101MHz,CDCl 3)δ173.86(d,J＝17.1Hz),140.66(s),138.76(s),134.93(s),129.74(s),129.24(s),126.19(s),77.36(s),77.04(s),76.72(s),65.01(s),64.48(s),57.73(s),55.73(s),53.93(s),34.76(s),34.28(d,J＝3.9Hz),33.54(d,J＝4.5Hz),32.41(s),31.95(d,J＝16.5Hz),29.49(s),29.15(dd,J＝21.1,2.4Hz),28.66(s),27.04(s),25.95(d,J＝3.3Hz), 24.85(d,J＝6.6Hz),22.98–22.58(m),14.08(d,J＝7.5Hz).
J.SW-II-135-1
Figure PCTCN2022131289-appb-000073
1、化合物3的合成
Figure PCTCN2022131289-appb-000074
化合物1(500mg,2.16mmol,1.0eq.)和化合物2(750mg,6.46mmol,3.0eq.)溶解在甲苯/H 2O(5mL/1mL)中，向该混合物中加入Ruphos(201mg,0.43mmol,0.2eq)，Pd(OAc) 2(48.5mg,0.22mmol,0.1eq)和Cs 2CO 3(2.10g,6.46mmol,3.0eq.)。反应混合物在氮气保护下110℃加热回流16小时。TLC(石油醚/乙酸乙酯＝10/1)显示反应完成并且形成了所需产物。反应混合物用H 2O(40mL)洗涤并用EA(50mL)萃取3次，所得有机相用盐水(20mL)洗涤两次，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-30/1)洗脱，得到黄色油状化合物3(540mg,82.44％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000075
在氮气保护0℃下，向溶解在THF(5mL)的化合物3(540mg,1.78mmol,1.0eq.)中加入LiAlH 4(3.55mL,3.55mmol,1M THF中,2eq.)。反应物升至室温氮气保护下搅拌2小时。TLC(PE/EtOAc＝10/1)显示反应完成并观察到新的主要斑点。混合物用水(10mL)淬灭，然后用1M盐酸调节pH＝6-7，用乙酸乙酯(50mL)萃取3次。有机层用盐水洗涤，经无水硫酸钠干燥，过滤，并减压浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-10/1)洗脱，得到无色油状的化合物4(442mg,90.2％)。
3、化合物6的合成
Figure PCTCN2022131289-appb-000076
将化合物4(442mg,1.60mmol,1.0eq.)和化合物5(428.5mg,1.92mmol,1.2eq.)溶解在DCM(5mL)中，向该混合物中加入EDCI(612mg,3.2mmol,2.0eq.)和DMAP(78.2mg,0.64mmol,0.4eq.)，然后加入DIEA(826mg,6.4mmol,4.0eq.)。反应混合物在氮气保护下 室温搅拌16小时。TLC(石油醚/乙酸乙酯＝10/1)显示化合物4被消耗并且形成了所需产物。反应混合物用H 2O(40mL)洗涤并用EA(50mL)萃取3次，所得有机相用盐水(20mL)洗涤两次，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-10/1)洗脱，得到黄色油状化合物3(342mg,44.5％)。
4、SW-II-135-1的合成
Figure PCTCN2022131289-appb-000077
化合物6(175mg,0.365mmol,1.2eq.)和化合物7(100mg,0.304mmol,1.0eq)溶解在CPME/CH 3CN(1mL/1mL)中，向该混合物中加入碳酸钾(210mg,1.52mmol,5.0eq)和碘化钾(101mg,0.61mmol,2.0eq)。添加完后，反应混合物在氮气保护下90℃搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完全并且形成了所需产物。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-135-1(83.89mg,55.6％)。
LCMS:Rt:1.356min；MS m/z(ELSD):730.5[M+H] +；
HPLC:100％purity at ELSD；RT＝12.614min.
1H NMR(400MHz,CDCl3)δ6.97(d,J＝7.6Hz,1H),6.91–6.74(m,2H),4.76(s,1H),3.99(dt,J＝13.6,6.4Hz,4H),3.72–3.58(m,2H),2.85–2.73(m,2H),2.72–2.61(m,4H),2.59–2.41(m,6H),2.22(dd,J＝13.2,7.2Hz,4H),1.93–1.79(m,2H),1.62–1.41(m,14H),1.23(d,J＝24.4Hz,32H),0.82(ddd,J＝13.6,8.0,5.6Hz,9H).
13C NMR(101MHz,CDCl3)δ172.81(d,J＝6.4Hz),139.55(s),137.38(s),137.14(s),128.16(d,J＝2.4Hz),124.69(s),76.51(s),76.19(s),75.88(s),63.43(s),62.78(s),56.53(s),54.90(s),52.84(s),33.23(d,J＝2.4Hz),31.73(s),31.28(s),30.91(dd,J＝20.0,6.4Hz),30.10(d,J＝3.2Hz),29.29(s),28.36(d,J＝22.8Hz),28.23(s),27.97(s),27.64(s),25.92(s),24.92(s),24.34(s),23.84(s),21.62(d,J＝7.6Hz),13.08(d,J＝4.7Hz).
K.化合物SW-II-135-2
Figure PCTCN2022131289-appb-000078
SW-II-135-2
1、化合物3的合成
Figure PCTCN2022131289-appb-000079
化合物1(500mg,2.16mmol,1.0eq.)和化合物2(931mg,6.46mmol,3.0eq.)溶解在甲苯/H 2O(5mL/1mL)中，向该混合物中加入Ruphos(201mg,0.43mmol,0.2eq)、Pd(OAc) 2(48.5mg,0.22mmol,0.1eq)和Cs 2CO 3(2.10g,6.46mmol,3.0eq.)。反应混合物在氮气保护下110℃加热回流16小时。TLC(石油醚/乙酸乙酯＝10/1)显示反应完成并且形成了所需产物。反应混合物用H 2O(40mL)洗涤并用EA(50mL)萃取3次，所得有机相用盐水(20mL)洗涤两次，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-30/1)洗脱，得到黄色油状化合物3(651mg,84％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000080
在氮气保护0℃下，向溶解在THF(7mL)的化合物3(651mg,1.81mmol,1.0eq.)中加入LiAlH 4(3.62mL,3.62mmol,1M，THF中,2eq.)。反应物升至室温氮气保护下搅拌2小时。TLC(PE/EtOAc＝10/1)显示反应完成并观察到新的主要斑点。混合物用水(10mL)淬灭，然后用1M盐酸调节pH＝6-7，用乙酸乙酯(50mL)萃取3次。有机层用盐水洗涤，经无水硫酸钠干燥，过滤，并减压浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-10/1)洗脱，得到无色油状的化合物4(571mg,95.2％)。
3、化合物6的合成
Figure PCTCN2022131289-appb-000081
将化合物4(571mg,1.72mmol,1.0eq.)和化合物5(459mg,2.06mmol,1.2eq.)溶解在DCM(6mL)中，向该混合物中加入EDCI(657mg,3.44mmol,2.0eq.)和DMAP(84mg,0.68mmol,0.4eq.)，然后加入DIEA(887.5mg,6.88mmol,4.0eq.)。反应混合物在氮气保护下室温搅拌16小时。TLC(石油醚/乙酸乙酯＝10/1)显示化合物4被消耗并且形成了所需产物。反应混合物用H 2O(50mL)洗涤并用EA(60mL)萃取3次，所得有机相用盐水(25mL)洗涤两次，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-10/1)洗脱，得到黄色油状化合物3(245mg,26.5％)。
4、SW-II-135-2的合成
Figure PCTCN2022131289-appb-000082
化合物6(245mg,0.456mmol,1.5eq.)和化合物7(100mg,0.3mmol,1.0eq)溶解在CPME/CH 3CN(1mL/1mL)中，向该混合物中加入碳酸钾(210mg,1.52mmol,5.0eq)和碘化钾(101mg,0.61mmol,2.0eq)。添加完后，反应混合物在氮气保护下90℃搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完全并且形成了所需产物。混合物用乙酸乙酯萃取并用水 洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-135-2(31.41mg,21.9％)。
LCMS:Rt:1.608min；MS m/z(ELSD):786.4[M+H] +；
HPLC:95.16％纯度，ELSD；RT＝7.919min.
1H NMR(400MHz,CDCl 3)δ6.98(d,J＝7.6Hz,1H),6.87(d,J＝2.4Hz,2H),4.28–4.13(m,1H),4.04–3.95(m,4H),3.94–3.84(m,2H),3.14–2.89(m,6H),2.59–2.43(m,6H),2.23(dd,J＝13.8,7.2Hz,4H),1.88–1.82(m,2H),1.70(s,4H),1.57–1.46(m,10H),1.33–1.16(m,40H),0.90–0.72(m,9H).
13C NMR(100MHz,CDCl 3)δ172.82(d,J＝6.8Hz),139.61(s),137.29(d,J＝16.4Hz),128.15(s),124.67(s),76.41(s),76.09(s),75.77(s),63.50(s),62.87(s),55.49(s),54.92(s),52.98(s),33.16(d,J＝2.4Hz),31.77(s),31.33(s),30.80(d,J＝6.5Hz),30.42(d,J＝3.6Hz),29.29(s),28.99–28.66(m),28.47(s),28.23(d,J＝2.8Hz),28.06–27.45(m),25.58(s),24.91(s),23.71(s),22.79(s),21.66(s),13.10(s).
L.化合物SW-II-136-2
Figure PCTCN2022131289-appb-000083
SW-II-136-2
1、化合物3的合成
Figure PCTCN2022131289-appb-000084
化合物1(3g,13.70mmol,1.0eq.)和化合物2(5.34g,41.09mmol,3.0eq.)溶解在甲苯/H 2O(30mL/3mL)中，向该混合物中加入Ruphos(1.28g,2.74mmol,0.2eq)，Pd(OAc) 2(308.3mg,1.37mmol,0.1eq)和K 2CO 3(5.67g,41.10mmol,3.0eq.)。反应混合物在氮气保护下110℃加热回流16小时。TLC(PE/EA＝10/1)显示反应完成并且形成了所需产物。反应混合物用H 2O(90mL)洗涤并用EA(110mL)萃取3次，所得有机相用盐水(40mL)洗涤两次，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-30/1)洗脱，得到黄色油状化合物3(1.98g,45.5％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000085
在氮气保护0℃下，向溶解在THF(20mL)的化合物3(1.98g,6.23mmol,1.0eq.) 中加入LiAlH 4(1M,12.45mL,2.0eq)。反应物升至室温氮气保护下搅拌2小时。TLC(PE/EtOAc＝10/1)显示反应完成并观察到新的主要斑点。混合物用H 2O(70mL)淬灭，然后用1M盐酸调节pH＝6-7，用EA(80mL)萃取3次。有机层用盐水洗涤，经无水Na 2SO 4干燥，过滤，并减压浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-10/1)洗脱，得到无色油状的化合物4(1.28g,71.1％)。
3、化合物7的合成
Figure PCTCN2022131289-appb-000086
在氮气保护0℃下，向溶解在DCM(9mL)的化合物4(900g,3.1mmol,1.0eq.)中加入DMSO(3.63g,51.72mmol,15eq)、TEA(1.25g,12.4mmol,4.0eq)和PySO 3(1.27g,7.97mmol,2.57eq)。将混合物在0℃下搅拌30分钟，然后升至室温氮气保护下下搅拌90分钟。然后向混合物中添加化合物6(4.74g,13.62mmol,3.0eq.)，反应混合物在氮气保护下25℃反应2小时。TLC(PE/EA＝10/1)显示反应完成并且形成了所需产物。反应混合物用H 2O(60mL)洗涤并用EA(70mL)萃取3次，所得有机相用盐水(40mL)洗涤两次，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-10/1)洗脱，得到黄色油状化合物7(345mg,27.9％)。
4、化合物8的合成
Figure PCTCN2022131289-appb-000087
将化合物7(340mg,0.95mmol,1.0eq.)和Pd/C(100mg)加入到MeOH(4ml)中，反应混合物在室温下在氢气保护下搅拌16h。TLC(PE/EA＝10/1)显示原料消耗完全，并生成了所需产物。反应混合物通过硅藻土过滤并用MeOH(40mL×2)洗涤，经无水Na 2SO 4干燥并减压浓缩滤液，获得淡黄色油状化合物8(298mg,88.2％)。
5、化合物9的合成
Figure PCTCN2022131289-appb-000088
在氮气保护0℃下，向溶解在THF(3mL)的化合物8(298mg,0.83mmol,1.0eq.)中加入LiAlH 4(1M,1.66mL,2.0eq)。反应物升至室温氮气保护下搅拌2小时。TLC(PE/EtOAc＝10/1)显示反应完成并观察到新的主要斑点。混合物用H 2O(20mL)淬灭，然后用1M盐酸调节pH＝6-7，用EA(30mL)萃取3次。有机层用盐水洗涤，经无水Na 2SO 4干燥，过滤，并减压浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-10/1)洗脱，得到无色油状的化合物9(254mg,98.3％)。
6、化合物11的合成
Figure PCTCN2022131289-appb-000089
将化合物9(254mg,0.80mmol,1.0eq.)和化合物10(214mg,0.96mmol,1.2eq.)溶解在DCM(3mL)中，向该混合物中加入EDCI(305.6mg,1.6mmol,2.0eq.)和DMAP(39mg,0.32mmol,0.4eq.)，然后加入DIEA(412.8mg,3.2mmol,4.0eq.)。反应混合物在氮气保护下室温搅拌16小时。TLC(PE/EA＝10/1)显示化合物9被消耗并且形成了所需产物。反应混合物用1M盐酸调节pH＝4-6，并用EA(30mL)萃取3次，所得有机相用盐水(15mL)洗涤两次，经无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-7/1)洗脱，得到黄色油状化合物11(210mg,50.5％)。
7、SW-II-136-2的合成
Figure PCTCN2022131289-appb-000090
化合物11(200mg,0.38mmol,1.2eq.)和化合物12(105mg,0.32mmol,1.0eq)溶解在CPME/CH 3CN(1.5mL/1.5mL)中，向该混合物中加入K 2CO 3(220.2mg,1.60mmol,5.0eq)和KI(106mg,0.64mmol,2.0eq)。添加完后，反应混合物在氮气保护下90℃搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完全并且形成了所需产物。混合物用EA萃取并用水洗涤。有机层用无水Na 2SO 4干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-136-2(208mg,90.4％)。
LCMS:Rt:2.146min；MS m/z(ELSD):773.3[M+H] +；
HPLC:99.49％纯度，ELSD；RT＝8.055min.
1H NMR(400MHz,CDCl 3)δ7.04(d,J＝7.6Hz,1H),6.92(d,J＝9.6Hz,2H),4.45(s,1H),4.06(dd,J＝12.0,5.2Hz,4H),3.64(t,J＝5.2Hz,2H),2.72(t,J＝5.2Hz,2H),2.65–2.50(m,10H),2.29(t,J＝7.6Hz,4H),1.69–1.48(m,18H),1.41–1.24(m,36H),0.95–0.78(m,9H).
13C NMR(101MHz,CDCl 3)δ173.86(d,J＝2.8Hz),140.48(s),139.24(s),138.01(s),129.13(d,J＝14.8Hz),125.67(s),77.37(s),77.05(s),76.73(s),64.45(s),64.23(s),57.88(s),55.91(s),53.94(s),35.07(s),34.29(d,J＝3.2Hz),32.79(s),32.35(s),31.82(d,J＝8.4Hz),31.38(s),29.50(d,J＝2.4Hz),29.16(dd,J＝18.0,2.0Hz),28.66(s),28.35(s),27.78(s),27.08(s),26.02(d,J＝17.2Hz),24.89(d,J＝1.6Hz),22.65(s),14.10(s).
M.化合物SW-II-137-1
Figure PCTCN2022131289-appb-000091
1、化合物3的合成
Figure PCTCN2022131289-appb-000092
化合物1(500mg,1.95mmol,1.0eq.),溶解在甲苯(5.0mL)中，然后加入化合物2(239mg,2.34mmol,1.2eq.)、Pd(PPh 3) 4(225mg,0.19mmol,0.1eq)、水(1mL)和K 2CO 3(808g,5.85mmol,3.0eq.)。氮气保护下110℃反应3小时。TLC(PE/EA＝5/1)显示原料已经反应完并且形成所要产物。反应加H 2O(70mL)，EA(80mL×3)萃取。合并有机相饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并且减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-5:1，v/v)洗脱得到无色油状化合物(320mg,70％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000093
化合物3(300mg,1.28mmol,1.0eq.)溶解在THF(4.0mL)中，氮气保护下0℃加LAH(97mg,2.56mmol,2.0eq)。然后室温下反应2小时。TLC(PE/EA＝10/1)显示原料反应完全并且生成要的产物。加HCl(1M,4mL)溶液和H 2O(10mL)淬灭，EA(50mL×3)萃取。有机相使用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并且减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-10/1，v/v)洗脱得到黄色油状化合物4(224mg,84.8％)。
3、化合物6的合成
Figure PCTCN2022131289-appb-000094
化合物4(90mg,0.47mmol,1.0eq.)溶解在DCM(3.0mL)中，添加化合物5(127mg,0.56mmol,1.2eq.)、EDCI(180mg,0.94mmol,2.0eq.)、DIEA(242mg,1.88mmol,4.0eq.)和DMAP(23mg,0.18mmol,0.4eq.)。然后，氮气保护下室温反应过夜。TLC(PE/EA＝20/1)显示原料已经反映完并且形成需要产物。用HCl(1M)溶液淬灭反应，调PH＝4～6，用EA(40mL×3)萃取。合并有机相并用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物使用硅胶柱纯化，用PE/EA(1/0-20/1，v/v)洗脱得到无色油状化合物6(90mg,48.6％)。
4、SW-II-137-1的合成
Figure PCTCN2022131289-appb-000095
化合物6(90mg,0.25mmol,1.0eq.)溶解在MeCN(2mL)中加入化合物7(110mg,0.25mmol,1.0eq)、KI(76mg,0.50mmol,2.0eq)、CPME(2mL)和K 2CO 3(157mg,1.25mmol,5.0eq)。氮气保护下90℃反应过夜。TLC(DCM/MeOH＝10/1)显示原料反应完全并且形成了需要的产物。用水(50mL)淬灭，EA(40mL×3)萃取。合并有机相并用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用DCM/MeOH(1/0-10/1,v/v)洗脱得到黄色油状化合物(98mg,52.12％，SW-II-137-1)。
LCMS:Rt:1.596min；MS m/z(ELSD):758.4[M+H] +；
HPLC:98.02％纯度，ELSD；RT＝5.993min.
1H NMR(400MHz,CDCl 3)δ7.02(d,J＝8.8Hz,4H),4.92–4.71(m,1H),4.01(t,J＝6.4Hz,2H),3.78(s,1H),3.55(t,J＝5.2Hz,2H),2.76–2.40(m,10H),2.21(dd,J＝15.6,7.7Hz,4H),1.95–1.80(m,2H),1.49(ddd,J＝24.4,15.8,6.2Hz,15H),1.34–1.13(m,37H),0.82(dt,J＝13.6,7.2Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.79(s),173.57(s),140.49(s),138.30(s),128.43(s),128.22(s),77.43(s),77.11(s),76.79(s),74.11(s),63.66(s),57.96(s),55.75(s),53.90(s),35.22(s),34.63(s),34.20(d,J＝11.6Hz),33.70(s),31.80(d,J＝11.2Hz),30.30(s),29.51(d,J＝2.8Hz),29.13(dd,J＝9.6,6.8Hz),27.12(d,J＝2.8Hz),26.29(s),25.31(s),24.97(d,J＝15.6Hz),22.66(s),22.37(s),14.02(d,J＝15.2Hz) .
N.化合物SW-II-137-2
Figure PCTCN2022131289-appb-000096
1、化合物3的合成
Figure PCTCN2022131289-appb-000097
化合物1(500mg,2.06mmol,1.0eq.),化合物2(286mg,2.47mmol,1.2eq.)，Pd(PPh 3) 4(119mg,0.1mmol,0.1eq)和K 2CO 3(851mg,6.21mmol,3.0eq.)溶解在甲苯(5.0mL)中，加水(0.5mL)。然后，氮气保护下110℃反应3小时。TLC(PE/EA＝5/1)显示原料反应完全并且形成了所要的化合物。用H 2O(70mL)淬灭反应，EA(80mL×3)萃取。用饱和食盐水(2×30mL)洗涤有机相，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用 (PE/EA＝5/1,v/v)洗脱得到无色油状化合物3(420mg,87.5％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000098
化合物3(420mg,1.78mmol,1.0eq.)溶解在THF(3.0mL)中，氮气保护下0℃滴加LAH(1M,7mL,2.0eq)。然后，室温反应2小时。TLC(PE/EA＝5/1)显示原料反应完并且形成了需要的产物。用HCl(1M,4mL)溶液和H 2O(10mL)淬灭，EA(50mL×3)萃取。有机相用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并且减压旋干。残留物用硅胶柱纯化，用(PE/EA＝5/1，v/v)洗脱得到无色油状化合物4(320mg,94％)。
3、化合物6的合成
Figure PCTCN2022131289-appb-000099
化合物4(320mg,1.55mmol,1.0eq.)溶解在DCM(4.0mL)中，加化合物5(416mg,1.86mmol,1.2eq.)，EDCI(594mg,3.11mmol,2.0eq.)，DIEA(802mg,6.21mmol,4.0eq.)和DMAP(76mg,0.62mmol,0.4eq.)。然后，氮气保护下室温反应过夜。TLC(PE/EA＝20/1)显示原料反应完并形成需要产物。反应用HCl(1M)溶液淬灭并调PH＝4～6，DCM(60mL×3)萃取。有机相用饱和食盐水(2×35mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用(PE/EA＝5/1,v/v)洗脱得到无色油状化合物6(300mg,47.17％)。
4、SW-II-137-2的合成
Figure PCTCN2022131289-appb-000100
化合物6(167mg,0.41mmol,1.2eq.)，化合物7(150mg,0.34mmol,1.0eq)，KI(113mg,0.68mmol,2.0eq)和CPME(2mL)溶解在MeCN(2mL)中，加K 2CO 3(235mg,1.70mmol,5.0eq)。氮气保护下90℃反应过夜。TLC(DCM/MeOH＝10/1)显示原料反应完全并且生成需要的产物。反应用水(50mL)淬灭，EA(60mlx3)萃取。有机相用无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用DCM/MeOH(1/0-10/1,v/v)洗脱得到浅黄色油状化合物(105mg,40.3％,SW-II-137-2)。
LCMS:Rt:1.660min；MS m/z(ELSD):772.4[M+H] +；
HPLC:98.38％纯度，ELSD；RT＝8.743min.
1H NMR(400MHz,CDCl 3)δ7.10(d,J＝8.8Hz,4H),5.04–4.74(m,1H),4.08(t,J＝6.4Hz,2H),3.58(t,J＝5.2Hz,2H),2.65(dd,J＝9.6,5.6Hz,4H),2.60–2.44(m,6H),2.29(dd,J＝16.4,7.6Hz,4H),2.01–1.88(m,2H),1.59(dt,J＝9.2,7.2Hz,6H),1.54–1.42(m, 8H),1.39–1.11(m,41H),0.88(dt,J＝11.8,6.0Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.86(s),173.63(s),140.59(s),138.34(s),128.45(s),128.24(s),77.36(s),77.04(s),76.72(s),74.14(s),63.69(s),58.11(s),55.71(s),53.90(s),35.53(s),34.68(s),34.23(d,J＝14.8Hz),31.82(d,J＝11.6Hz),31.56(s),31.26(s),30.32(s),29.53(d,J＝2.8Hz),29.19(dd,J＝8.0,4.4Hz),27.20(d,J＝2.4Hz),26.64(s),25.33(s),25.02(d,J＝15.6Hz),22.62(d,J＝11.6Hz),14.08(d,J＝8.0Hz).
O.化合物SW-II-137-3
Figure PCTCN2022131289-appb-000101
1、化合物3的合成
Figure PCTCN2022131289-appb-000102
向化合物1(11.8g,53mmol,1.2eq.)和化合物2(11.2g,44mmol,1eq.)在DCM(110mL)中的混合物中加入EDCI(16.9g,88mmol,2eq.)和DMAP(2.1g,18mmol,0.4eq.)，然后加入DIEA(22.7g,176mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝30/1)显示化合物1被消耗并且形成了所需产物。反应混合物用乙酸乙酯(200mL)萃取并用水(200mL×3)洗涤，经无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到无色油状化合物3(7.391g,37％)。
2、化合物5的合成
Figure PCTCN2022131289-appb-000103
将化合物3(7.391mg,16.07mmol,1eq.)和化合物4(29.4g,482.02mmol,30eq.)在乙醇(2mL)中的混合物在55℃下在氮气下搅拌16小时。TLC(DCM/MeOH＝10/1)显示观察到新的主要斑点。反应混合物用乙酸乙酯(100mL)萃取并用水(3×100mL)洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1，v/v)洗脱，得到黄色油状化合物5(3.695g,52％)。
3、化合物8的合成
Figure PCTCN2022131289-appb-000104
向化合物6(1g,4.12mmol,1eq.)和化合物7(803g,6.17mmol,1.5eq)在1,4-二氧六环/水(10mL/1mL)中的混合物中加入Pd(dtbpf)Cl 2(269mg,0.41mmol,0.1eq.)和碳酸钾(1.7g,12.36mmol,3eq)。将混合物在氮气下于100℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物8(568mg,56％)。
4、化合物9的合成
Figure PCTCN2022131289-appb-000105
在0℃和氮气环境下，向化合物8(568mg,2.29mmol,1eq.)在THF(6mL)中的混合物中加入氢化铝锂(2.3mL,2.29mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(2.3mL)淬灭并用2N盐酸处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物9(541mg,>100％)，无需进一步纯化。
5、化合物10的合成
Figure PCTCN2022131289-appb-000106
向化合物9(441mg,2mmol,1eq.)和化合物1(536mg,2.4mmol,1.2eq.)在DCM(5mL)中的混合物中加入EDCI(768mg,4mmol,2eq.)和DMAP(98mg,0.8mmol,0.4eq.)，然后加入DIEA(1.032g,8mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝10/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-10/1)洗脱，得到无色油状化合物10(372mg,44％)。
6、SW-II-137-3的合成
Figure PCTCN2022131289-appb-000107
向化合物10(150mg,0.353mmol,1eq.)和化合物5(156mg,0.353mmol,1eq.)在 CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(244mg,1.765mmol,6eq.)和碘化钾(117mg,0.706mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-137-3(56.17mg,20％)。
LCMS:Rt:1.550min；MS m/z(ELSD):786.4[M+H] +；
HPLC:98.597％纯度，ELSD；RT＝13.153min.
1H NMR(400MHz,CDCl 3)δ7.09(s,4H),4.92–4.78(m,1H),4.08(t,J＝6.6Hz,2H),3.62(t,J＝5.2Hz,2H),2.78–2.50(m,10H),2.35–2.22(m,4H),2.00–1.88(m,2H),1.57(ddd,J＝28.9,13.5,4.5Hz,14H),1.38–1.20(m,42H),0.88(t,J＝6.8Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.83(s),173.60(s),140.60(s),138.33(s),128.44(s),128.24(s),77.36(s),77.04(s),76.72(s),74.15(s),63.69(s),57.95(s),55.83(s),53.95(s),35.56(s),34.65(s),34.21(d,J＝13.0Hz),31.81(d,J＝12.3Hz),31.54(s),30.32(s),29.52(d,J＝3.1Hz),29.34–28.94(m),27.13(d,J＝2.5Hz),26.31(s),25.33(s),24.99(d,J＝15.7Hz),22.64(d,J＝5.7Hz),14.11(s).
P.化合物SW-II-138-1
Figure PCTCN2022131289-appb-000108
1、化合物2的合成
Figure PCTCN2022131289-appb-000109
化合物1(4g,16.46mmol,1.0eq.)溶解在MeOH(40mL)中，冷却至0℃滴加SOCl 2(3.9g,32.92mmol,2.0eq)。然后室温反应1小时。TLC(PE/EA＝5/1)显示原料消耗完并形成了需要产物。体系直接减压旋干，残留物中加入NaHCO 3(70mL)溶液，用EA(80mL×3)萃取。合并有机相并用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-5:1，v/v)洗脱得到黄色油状化合物2(4.1mg,95％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000110
化合物2(500mg,1.95mmol,1.0eq.),化合物3(239mg,2.34mmol,1.2eq.)、Pd(PPh 3) 4(225mg,0.19mmol,0.1eq)和K 2CO 3(808g,5.85mmol,3.0eq.)溶解在甲苯(5.0mL)中加水 (1mL)。然后氮气保护下110℃反应3小时。TLC(PE/EA＝5/1)显示原料消耗完并形成了需要的产物。反应加H 2O(70mL)淬灭，EA(80mL×3)萃取。合并有机相并用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物使用硅胶柱纯化，用PE/EA(1/0-5:1，v/v)洗脱得到黄色油状化合物4(320mg,70％)。
3、化合物5的合成
Figure PCTCN2022131289-appb-000111
化合物4(300mg,1.28mmol,1.0eq.)溶解在THF(4.0mL)中，0℃加LAH(97mg,2.56mmol,2.0eq)。然后氮气保护下室温反应2小时。TLC(PE/EA＝5/1)显示原料消耗完并形成了需要的产物。加HCl(1M,4mL)溶液和H 2O(10mL)淬灭反应,用EA(50mL×3)萃取。有机相用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-5:1，v/v)洗脱得到黄色油状化合物5(224mg,84.8％)。
4、化合物7的合成
Figure PCTCN2022131289-appb-000112
化合物7(224mg,1.09mmol,1.0eq.)溶解在DCM(3.0mL)中，加化合物6(290mg,1.30mmol,1.2eq.)，EDCI(415mg,2.17mmol,2.0eq.)、DIEA(561mg,4.35mmol,4.0eq.)和DMAP(53mg,0.43mmol,0.4eq.)。然后，氮气保护下室温反应过夜。TLC(PE/EA＝30/1)显示原料消耗完并形成了需要的产物。反应用HCl(1M)溶液淬灭并调节PH＝4～6，用DCM(80mL×3)萃取。合并的有机相用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-30:1，v/v)洗脱得到无色油状化合物7(208mg,46.7％)。
5、SW-II-138-1的合成
Figure PCTCN2022131289-appb-000113
化合物10(110mg,0.25mmol,1eq.)、化合物7(153mg,0.37mmol,1.5eq)、KI(83mg、0.50mmol,2.0eq)和CPME(2mL)溶解在MeCN(2mL)中加K 2CO 3(172mg,1.25mmol,5.0eq)。氮气保护下90℃反应过夜。TLC(DCM/MeOH＝10/1)显示原料消耗完并形成了需要的产物。反应直接减压旋干。残留物用硅胶柱纯化，用DCM/MeOH(1/0-10:1，v/v)洗脱得到浅黄色油状化合物(65mg,32％，SW-II-138-1)。
LCMS:Rt:1.684min；MS m/z(ELSD):772.4[M+H] +；
HPLC:96.56％纯度，ELSD；RT＝6.346min.
1H NMR(400MHz,CDCl 3)δ7.09(s,4H),4.86(s,1H),4.09(d,J＝6.0Hz,2H),3.97(s, 2H),3.07(d,J＝38.8Hz,6H),2.69–2.51(m,4H),2.28(td,J＝7.3,3.6Hz,4H),1.79(s,4H),1.70–1.46(m,16H),1.42–1.17(m,37H),0.90(dt,J＝13.6,7.2Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.80(s),173.53(s),140.32(s),139.13(s),128.28(d,J＝13.6Hz),77.43(s),77.11(s),76.80(s),74.21(s),64.22(s),56.85(s),55.98(s),53.93(s),35.22(s),35.01(s),34.54(s),34.14(d,J＝5.6Hz),33.71(s),31.85(s),29.50(d,J＝2.8Hz),29.22(s),29.12–28.60(m),28.26(s),27.78(s),26.70(d,J＝4.4Hz),25.31(s),24.82(d,J＝17.6Hz),24.28(s),22.65(s),22.37(s),14.03(d,J＝15.2Hz).
Q.SW-II-138-2
Figure PCTCN2022131289-appb-000114
1、化合物3的合成
Figure PCTCN2022131289-appb-000115
化合物1(500mg,1.95mmol,1.0eq.),化合物2(271mg,2.34mmol,1.2eq.)、Pd(PPh 3) 4(225mg,0.20mmol,0.1eq)和K 2CO 3(809g,5.86mmol,3.0eq.)溶解在甲苯(5.0mL)中加水(1mL)，然后氮气保护下110℃反应3小时。TLC(PE/EA＝5/1)显示原料消耗完并形成了需要的产物。反应加水(70mL)淬灭，用EA(80mL×3)萃取。合并有机相并用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-30:1，v/v)洗脱得到无色油状化合物3(320mg,70％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000116
化合物3(320mg,1.29mmol,1.0eq.)溶解在THF(3.0mL)中，0℃加LAH(67mg,1.77mmol,2.0eq),然后，氮气保护下室温反应2小时。TLC(PE/EA＝5/1)显示原料消耗完并形成了需要的产物。反应用HCl(1M,2mL)溶液和H 2O(10mL)淬灭，EA(50mL×3)萃取。合并的有机相用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-30:1，v/v)洗脱得到无色油状化合物4(180mg,64％)。
3、化合物6的合成
Figure PCTCN2022131289-appb-000117
化合物4(180mg,0.82mmol,1.0eq.)溶解在DCM(3.0mL)中加化合物5(245mg, 1.10mmol,1.2eq.)、EDCI(347mg,1.82mmol,2.0eq.)、DIEA(470mg,3.63mmol,4.0eq.)和DMAP(45mg,0.36mmol,0.4eq.)。然后，反应在氮气保护下室温过夜。TLC(PE/EA＝30/1)显示原料消耗完全并形成了需要的产物。反应用HCl(1M)溶液淬灭并调节PH＝5～6，用DCM(80mL×3)萃取。合并的有机相用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-30:1,v/v)洗脱得到无色油状化合物6(220mg,63.6％)。
4、SW-II-138-2的合成
Figure PCTCN2022131289-appb-000118
化合物6(158mg,0.37mmol,1.5eq.)和化合物7(110mg,0.25mmol,1.0eq)、KI(83mg,0.50mmol,2.0eq)和CPME(2mL)溶解在MeCN(2mL)中，加K 2CO 3(172mg,1.25mmol,5.0eq)。然后，反应在氮气保护下90℃反应过夜。TLC(DCM/MeOH＝10/1)显示原料消耗完并形成了需要的产物。反应直接减压旋干，残留物用硅胶柱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱得到无色油状目标产物(100mg,51％,SW-II-138-2)。
LCMS:Rt:1.834min；MS m/z(ELSD):786.4[M+H] +；
HPLC:99.20％纯度，ELSD；RT＝7.990min.
1H NMR(400MHz,CDCl 3)δ7.00(s,4H),4.88–4.73(m,2H),4.00(t,J＝5.6Hz,2H),3.81–3.54(m,2H),3.00–2.81(m,2H),2.81–2.65(m,4H),2.50(dd,J＝16.4,8.4Hz,4H),2.20(td,J＝7.6,3.2Hz,4H),1.56(ddd,J＝18.4,10.4,5.2Hz,13H),1.43(d,J＝5.6Hz,4H),1.34–1.07(m,40H),0.81(dt,J＝11.2,5.6Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.78(s),173.52(s),140.32(s),139.11(s),128.25(d,J＝11.6Hz),77.49(s),77.17(s),76.85(s),74.14(s),64.17(s),57.25(s),55.82(s),53.85(s),35.50(s),35.01(s),34.56(s),34.14(d,J＝7.2Hz),31.84(s),31.53(s),31.23(s),29.49(d,J＝2.8Hz),29.21(s),28.94(dd,J＝6.4,4.4Hz),28.25(s),27.77(s),26.84(d,J＝4.4Hz),25.30(s),25.25–24.59(m),22.59(d,J＝11.2Hz),14.05(d,J＝7.6Hz).
R.SW-II-138-3
Figure PCTCN2022131289-appb-000119
1、化合物3的合成
Figure PCTCN2022131289-appb-000120
化合物1(500mg,1.95mmol,1.0eq.)、化合物2(305mg,2.34mmol,1.2eq.)、Pd(PPh 3) 4(225mg,0.20mmol,0.1eq)和K 2CO 3(809g,5.86mmol,3.0eq.)溶解在甲苯(5.0mL)中加水(1mL)。然后，反应在氮气保护下110℃反应3小时。TLC(PE/EA＝5/1)显示原料消耗完全并且形成要的化合物。反应加水(80mL)淬灭，用EA(80mL×3)萃取。合并有机相并用饱和食盐水(2×40mL)洗涤，无水Na 2SO 4干燥，过滤并且减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-5:1，v/v)洗脱得到无色油状化合物3(260mg,51.3％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000121
化合物3(260mg,0.99mmol,1.0eq.)溶解在THF(4.0mL)中，0℃加LAH(75mg,1.98mmol,2.0eq)。然后，氮气保护下室温反应2小时。TLC(PE/EA＝5/1)显示原料反应完并形成了需要的化合物。反应用HCl(1M,4mL)溶液和H 2O(20mL)淬灭，EA(50mL×3)萃取。合并有机相用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-5:1,v/v)洗脱得到无色油状化合物4(230mg,98％).
3、化合物6的合成
Figure PCTCN2022131289-appb-000122
化合物4(240mg,1.03mmol,1.0eq.)溶解在DCM(4.0mL)中，依次加入化合物5(275mg,1.23mmol,1.2eq.)、EDCI(392mg,2.07mmol,2.0eq.)、DIEA(530mg,4.10mmol,4.0eq.)和DMAP(50mg,0.41mmol,0.4eq.)。然后氮气保护下室温反应过夜。TLC(PE/EA＝20/1)显示原料消耗完并且形成了需要的化合物。反应用HCl(1M)淬灭并且调节PH＝5～6，DCM(80mL×3)萃取。合并的有机相用饱和食盐水(2×30mL)洗涤，无水Na 2SO 4干燥，过滤并且减压旋干。残留物用硅胶柱纯化，用PE/EA(1/0-20:1，v/v)洗脱得到无色油状化合物6(180mg,40.9％)。
4、SW-II-138-3的合成
Figure PCTCN2022131289-appb-000123
化合物6(164mg,0.37mmol,1eq.)和化合物7(110mg,0.24mmol,1.0eq)、KI(83mg,0.49mmol,2.0eq)和CPME(2mL)溶解在MeCN(2mL)中加入K 2CO 3(172mg,1.24mmol,5.0eq)。然后，氮气保护下90℃反应过夜。TLC(DCM/MeOH＝10/1)显示原料消耗完并且形成要的产物。反应直接减压旋干。残留物用硅胶柱纯化，用DCM/MeOH(1/0-10:1， v/v)洗脱得到无色油状目标产物(108mg,52.76％，SW-II-138-3)。
LCMS:Rt:2.007min；MS m/z(ELSD):800.4[M+H] +；
HPLC:97.95％纯度，ELSD；RT＝9.455min.
1H NMR(400MHz,CDCl 3)δ7.08(s,4H),4.86(p,J＝6.4Hz,1H),4.08(s,2H),3.60(t,J＝5.2Hz,3H),2.76–2.42(m,10H),2.28(td,J＝7.6,2.8Hz,4H),1.70–1.42(m,18H),1.28(d,J＝20.0Hz,41H),0.88(t,J＝6.8Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.85(s),173.59(s),140.39(s),139.15(s),128.27(d,J＝12.0Hz),77.38(s),77.07(s),76.75(s),74.13(s),64.17(s),58.06(s),55.75(s),53.92(s),35.57(s),35.03(s),34.66(s),34.22(d,J＝13.2Hz),31.81(d,J＝12.4Hz),31.54(s),29.52(d,J＝2.9Hz),29.34–28.95(m),28.29(s),27.79(s),27.16(d,J＝3.6Hz),26.50(s),25.32(s),24.99(d,J＝17.6Hz),22.64(d,J＝5.6Hz),14.10(s).
S.化合物SW-II-139-1
Figure PCTCN2022131289-appb-000124
1、化合物3的合成
Figure PCTCN2022131289-appb-000125
向化合物1(1g,4.37mmol,1eq.)和化合物2(852g,6.55mmol,1.5eq)在1,4-二氧六环/水(10mL/1mL)中的混合物中加入Pd(dtbpf)Cl 2(286mg,0.437mmol,0.1eq.)和碳酸钾(1.8g,13.11mmol,3eq)。将混合物在氮气下于100℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物3(691mg,68％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000126
在0℃和氮气环境下，向化合物3(691mg,2.95mmol,1eq.)在THF(7mL)中的混合物中加入氢化铝锂(3mL,2.95mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(3mL)淬灭并用2N盐酸处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物4(547mg,90％)，无需进一步纯化。
3、化合物6的合成
Figure PCTCN2022131289-appb-000127
向化合物4(447mg,2.17mmol,1eq.)和化合物5(581mg,2.6mmol,1.2eq.)在DCM(5mL)中的混合物中加入EDCI(833mg,4.34mmol,2eq.)和DMAP(106mg,0.87mmol,0.4eq.)，然后加入DIEA(1.12g,8.68mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝15/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到无色油状化合物6(455mg,51％)。
4、SW-II-139-1的合成
Figure PCTCN2022131289-appb-000128
向化合物6(150mg,0.365mmol,1eq.)和化合物7(161mg,0.365mmol,1eq.)在CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(252mg,1.825mmol,6eq.)和碘化钾(121mg,0.73mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1，v/v)洗脱，得到黄色油状化合物SW-II-139-1(54.53mg,19％)。
LCMS:Rt:1.521min；MS m/z(ELSD):772.4[M+H] +；
HPLC:99.637％纯度，ELSD；RT＝12.347min.
1H NMR(400MHz,CDCl 3)δ7.20(t,J＝7.7Hz,1H),7.03(t,J＝6.8Hz,3H),4.94–4.78(m,1H),4.27(t,J＝7.2Hz,2H),3.65(t,J＝5.1Hz,2H),2.90(t,J＝7.2Hz,2H),2.73(t,J＝4.9Hz,2H),2.67–2.41(m,6H),2.28(td,J＝7.5,2.7Hz,4H),1.67–1.45(m,14H),1.41–1.19(m,42H),0.88(dd,J＝7.9,5.7Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.65(d,J＝11.3Hz),143.17(s),137.67(s),129.04(s),128.34(s),126.61(s),126.11(s),77.30(d,J＝11.6Hz),77.04(s),76.72(s),74.16(s),64.85(s),57.88(s),55.93(s),53.97(s),35.94(s),35.13(s),34.64(s),34.20(d,J＝10.5Hz),31.80(d,J＝13.7Hz),31.50(s),29.52(d,J＝2.9Hz),29.34–28.92(m),27.08(d,J＝3.9Hz),26.10(s),25.33(s),25.05(s),24.82(s),22.64(d,J＝6.5Hz),14.11(s).
T.化合物SW-II-139-2
Figure PCTCN2022131289-appb-000129
1、化合物3的合成
Figure PCTCN2022131289-appb-000130
向化合物1(1g,4.37mmol,1eq.)和化合物2(668g,6.55mmol,1.5eq)在1,4-二氧六环/水(10mL/1mL)中的混合物中加入Pd(dtbpf)Cl 2(286mg,0.437mmol,0.1eq.)和碳酸钾(1.8g,13.11mmol,3eq)。将混合物在氮气下于100℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物3(605mg,67％)油。
2、化合物4的合成
Figure PCTCN2022131289-appb-000131
在0℃和氮气环境下，向化合物3(605mg,2.94mmol,1eq.)在THF(7mL)中的混合物中加入氢化铝锂(3mL,2.94mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(3mL)淬灭并用2N盐酸处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物4(534mg,>100％)，无需进一步纯化。
3、化合物6的合成
Figure PCTCN2022131289-appb-000132
向化合物4(434mg,2.44mmol,1eq.)和化合物5(652mg,2.93mmol,1.2eq.)在DCM(5mL)中的混合物中加入EDCI(937mg,4.88mmol,2eq.)和DMAP(119mg,0.976mmol,0.4eq.)，然后加入DIEA(1.259g,9.76mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝15/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到无色油状化合物6(355mg,38％)。
4、SW-II-139-2的合成
Figure PCTCN2022131289-appb-000133
向化合物6(122mg,0.319mmol,1eq.)和化合物7(140mg,0.319mmol,1eq.)在CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(220mg,1.595mmol,5eq.)和碘化钾(106mg,0.638mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-139-2(45.48mg,19％)。
LCMS:Rt:1.346min；MS m/z(ELSD):744.3[M+H] +；
HPLC:97.994％纯度，ELSD；RT＝11.235min.
1H NMR(400MHz,CDCl 3)δ7.20(t,J＝7.8Hz,1H),7.03(t,J＝7.6Hz,3H),4.91–4.81(m,1H),4.27(t,J＝7.2Hz,2H),3.89–3.75(m,2H),2.99–2.79(m,7H),2.64–2.48(m,2H),2.28(td,J＝7.5,3.1Hz,4H),1.74–1.08(m,53H),0.90(dt,J＝13.6,7.2Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.60(d,J＝11.7Hz),143.13(s),137.65(s),129.06(s),128.34(s),126.64(s),126.11(s),77.30(d,J＝11.4Hz),77.04(s),76.72(s),74.22(s),64.88(s),57.28(s),56.55(s),54.11(s),35.60(s),35.12(s),34.56(s),34.15(d,J＝4.0Hz),33.68(s),31.86(s),29.52(d,J＝2.8Hz),29.24(s),28.91(dd,J＝7.0,4.2Hz),26.81(d,J＝3.9Hz),25.33(s),25.12–24.98(m),24.83(d,J＝22.2Hz),22.67(s),22.40(s),14.04(d,J＝14.4Hz).
U.化合物SW-II-140-1
Figure PCTCN2022131289-appb-000134
1、化合物3的合成
Figure PCTCN2022131289-appb-000135
向化合物1(1g,4.37mmol,1eq.)和化合物2(852g,6.55mmol,1.5eq)在1,4-二氧六环/水(10mL/1mL)中的混合物中加入Pd(dppf)Cl 2(286mg,0.437mmol,0.1eq.)和碳酸钾(1.8g, 13.11mmol,3eq)。将混合物在氮气下于100℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物3(748mg,73％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000136
在0℃和氮气环境下，向化合物3(748mg,3.2mmol,1eq.)在THF(8mL)中的混合物中加入氢化铝锂(3.2mL,3.2mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(3mL)淬灭并用2N盐酸处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物4(493mg,75％)，无需进一步纯化。
3、化合物6的合成
Figure PCTCN2022131289-appb-000137
向化合物4(393mg,1.91mmol,1eq.)和化合物5(511mg,2.29mmol,1.2eq.)在DCM(5mL)中的混合物中加入EDCI(733mg,3.82mmol,2eq.)和DMAP(93mg,0.76mmol,0.4eq.)，然后加入DIEA(986mg,7.64mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝15/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到化合物6(327mg,42％)，为无色油。
4、SW-II-140-1的合成
Figure PCTCN2022131289-appb-000138
向化合物6(150mg,0.365mmol,1eq.)和化合物7(161mg,0.365mmol,1eq.)在CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(302mg,2.19mmol,6eq.)和碘化钾(121mg,0.73mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-140-1(180mg,64％)。
LCMS:Rt:1.568min；MS m/z(ELSD):772.4[M+H] +；
HPLC:98.053％纯度，ELSD；RT＝8.702min.
1H NMR(400MHz,CDCl 3)δ7.23–7.05(m,4H),4.95–4.79(m,1H),4.25(t,J＝7.4Hz,2H),3.62(t,J＝4.8Hz,2H),2.96(dd,J＝15.4,8.0Hz,2H),2.74–2.49(m,8H),2.28(dd,J＝14.2,7.2Hz,4H),1.67–1.44(m,14H),1.41–1.20(m,42H),0.90(dt,J＝13.2,7.1Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.68(d,J＝10.2Hz),141.26(s),135.23(s),129.73(s),129.37(s),126.72(s),125.92(s),77.35(s),77.03(s),76.71(s),74.17(s),64.52(s),57.99(s),55.87(s),53.94(s),34.66(s),34.21(d,J＝11.5Hz),32.75(s),31.83(d,J＝9.8Hz),31.32(s),29.65–28.88(m),27.15(d,J＝3.7Hz),26.35(s),25.33(s),25.07(s),24.83(s),22.66(d,J＝3.4Hz),14.12(s).
V.SW-II-140-2
Figure PCTCN2022131289-appb-000139
1、化合物3的合成
Figure PCTCN2022131289-appb-000140
向化合物1(1g,4.37mmol,1eq.)和化合物2(668g,6.55mmol,1.5eq)在1,4-二氧六环/水(10mL/1mL)中的混合物中加入Pd(dppf)Cl 2(286mg,0.437mmol,0.1eq.)和碳酸钾(1.8g,13.11mmol,3eq)。将混合物在氮气下于100℃搅拌过夜。TLC(PE/EA＝20/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用PE/EA(1/0-20/1)洗脱，得到无色油状化合物3(406mg,45％)。
2、化合物4的合成
Figure PCTCN2022131289-appb-000141
在0℃和氮气环境下，向化合物3(406mg,1.97mmol,1eq.)在THF(5mL)中的混合物中加入氢化铝锂(2mL,1.97mmol,1M,THF中,1eq.)。将混合物在室温下搅拌3小时。TLC(PE/EA＝5/1)表明反应完成并观察到新的主要斑点。混合物用水(2mL)淬灭并用2N盐酸 处理以将PH调节在6和7之间，用乙酸乙酯萃取并用盐水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩，得到无色油状化合物4(341mg,97％)，无需进一步纯化。
3、化合物6的合成
Figure PCTCN2022131289-appb-000142
向化合物4(241mg,1.35mmol,1eq.)和化合物5(361mg,1.62mmol,1.2eq.)在DCM(3mL)中的混合物中加入EDCI(518mg,2.7mmol,2eq.)和DMAP(66mg,0.54mmol,0.4eq.)，然后加入DIEA(697mg,5.4mmol,4eq.)。将反应混合物在室温下在氮气下搅拌16小时。TLC(石油醚/乙酸乙酯＝15/1)显示形成了所需产物。反应混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并真空浓缩。残余物通过硅胶柱色谱纯化，用石油醚/乙酸乙酯(1/0-20/1)洗脱，得到无色油状化合物6(185mg,32％)。
4、SW-II-140-2的合成
Figure PCTCN2022131289-appb-000143
向化合物6(185mg,0.483mmol,1eq.)和化合物7(213mg,0.483mmol,1eq.)在CPME/CH 3CN(2mL/2mL)中的混合物中加入碳酸钾(400mg,2.898mmol,6eq.)和碘化钾(160mg,0.966mmol,2eq.)。添加后，将混合物在氮气下在90℃下搅拌过夜。TLC(DCM/MeOH＝10/1)显示反应完成并观察到新的主要斑点。混合物用乙酸乙酯萃取并用水洗涤。有机层用无水硫酸钠干燥，过滤并减压浓缩。残余物通过硅胶柱色谱纯化，用DCM/MeOH(1/0-10:1,v/v)洗脱，得到黄色油状化合物SW-II-140-2(161mg,45％)。
LCMS:Rt:1.696min；MS m/z(ELSD):744.3[M+H] +；
HPLC:94.658％纯度，ELSD；RT＝5.938min.
1H NMR(400MHz,CDCl 3)δ7.22–7.03(m,4H),4.94–4.78(m,1H),4.25(t,J＝7.3Hz,2H),3.70–3.54(m,2H),2.96(t,J＝7.4Hz,2H),2.77–2.41(m,8H),2.28(dd,J＝14.3,7.1Hz,4H),1.65–1.18(m,52H),0.91(dt,J＝13.3,7.1Hz,9H).
13C NMR(101MHz,CDCl 3)δ173.67(d,J＝10.8Hz),141.22(s),135.23(s),129.73(s),129.39(s),126.72(s),125.92(s),77.36(s),77.04(s),76.72(s),74.17(s),64.52(s),57.92(s),55.92(s),53.96(s),34.66(s),34.21(d,J＝11.2Hz),33.51(s),32.44(s),31.83(d,J＝9.3Hz),29.53(d,J＝2.9Hz),29.14(dd,J＝11.3,8.5Hz),27.12(d,J＝4.1Hz),26.23(s),25.33(s),25.06(s),24.82(s),22.73(d,J＝9.9Hz),14.08(d,J＝8.8Hz).
实施例2.包含不同3'-UTR元件的质粒及内参对照质粒的基因克隆
包含不同3'-UTR元件的质粒为：通过同源重组的基因手段将不同的3'-UTR元件(序 列如SEQ ID NO:1-43所示)克隆到报告基因萤火虫荧光素酶(Firefly Luciferase)的下游终止密码子与poly(A)序列的中间区域，即如图1所示的3'-UTR区域，其余部分相同。将萤火虫荧光素酶下游终止密码子后仅含poly(A)序列，不含测试的3'-UTR元件的质粒作为参比构建体。图2为示例性的受试人工核酸分子(SEQ ID NO:99，其中示例性的3'-UTR元件为SEQ ID NO:1)，下划线处为测试的3'-UTR元件。除了所述下划线的元件之外，图2中显示的该序列的所有元件与参比人工核酸分子(SEQID NO:100)相同。因此，SEQ ID NO:99与SEQ ID NO:100的区别仅在于在poly(A)序列之前有测试的不同的3'-UTR元件。
内参对照质粒为：将报告基因萤火虫荧光素酶(Firefly Luciferase)替换为海肾荧光素酶基因(renilla luciferase，Rluc)，质粒的其他序列均相同。
由上海生工生物工程公司全基因合成包含不同3'-UTR元件的质粒和内参对照质粒。
利用一对引物(上游通用引物：5’TTGGACCCTCGTACAGAAGCTAATACG 3’；和下游携带poly(T)的特异性互补长引物)和基于高保真DNA聚合酶的PCR扩增试剂盒(诺唯赞生物技术股份有限公司)进行PCR扩增获得DNA模板。
实施例3.mRNA的制备
以如实施例2制备的纯化(Takara纯化试剂盒)后的PCR产物为模板，利用T7RNA聚合酶进行共转录加帽反应，进行RNA的体外转录，从而产生Cap1mRNA。体外转录中加入1-甲基-假尿苷-三磷酸代替三磷酸尿苷(UTP)，因此，体外转录的Cap1mRNA中1-甲基-假尿嘧啶的修饰比例为100％。转录结束后，使用DNaseI(赛默飞世尔科技有限公司)消化DNA模板，以降低残余DNA模板带来的风险。使用DynabeadsMyone(赛默飞世尔科技有限公司)对mRNA进行纯化。将纯化的mRNA溶解于1mM柠檬酸钠缓冲液(pH 6.5+/-0.1)中，无菌过滤，并在-80℃下冷冻保存直至使用。
实施例4.候选3'-UTR元件对报告基因影响的细胞表达验证
将生长状态良好的HEK-293细胞接种到96孔细胞培养板中，接种密度为3.5×10 4个细胞/孔，然后置于37℃细胞培养箱中培养18-24小时。使用Lipofectamine Messenger MAX试剂(Thermo Fisher)，以每孔2:1的mRNA质量比同时转染实施例3含有报告基因萤火虫荧光素酶的质粒的mRNA和含有报告基因海参荧光素酶的内参对照质粒的mRNA(总计100ng mRNA)至96孔板的HEK293细胞中，每个样品设置三个复孔。将转染mRNA后的细胞板置于37℃、5％CO 2细胞培养箱中培养24小时。
使用双荧光素酶报告基因检测试剂盒(诺唯赞，DL101-01)对转染后的细胞样品进行检测。首先进行前处理，按照100μL/孔将细胞裂解液加入96孔细胞板中。然后，小心吸取14μL裂解后的细胞上清液，转移至96孔全黑酶标板中，再将70μL平衡至室温的萤火虫荧光素酶底物加入酶标板中，迅速混匀后立即使用酶标仪(BioTek)，振板10秒后， 在560nm波长处检测萤火虫荧光素酶的相对光单位(RLU)值；检测完毕后，在以上反应液中继续加入70μL新鲜配制的海肾荧光素酶底物工作液，迅速混匀后立即使用酶标仪(BioTek)，在480nm波长处检测海肾荧光素酶的相对光单位(RLU)值。每孔实际报告为每个孔中萤火虫荧光素酶RLU(Fluc)与海肾荧光素酶RLU(Rluc)的比值(Fluc/Rluc)。数据分析时，以仅含poly(A)序列的人工核酸分子的Fluc/Rluc为参比核酸分子，包含测试的不同的3'-UTR元件的人工核酸分子的Fluc/Rluc比值相对于参比的比值为最终的相对表达量。根据三次独立重复实验的结果分析不同3'-UTR元件对报告基因表达的影响，绘制统计直方图，并进行统计学分析。
实验结果如图3以及表2所示，大部分包含测试的3'-UTR元件的人工核酸分子的翻译效率高于参比核酸分子的翻译效率。其中，包含编号为U3006、U3008、U3009、U3010、U3011、U3016、U3020、U3030、U3051、U3053、U3055、U3056、U3057、U3058、U3060和U3067的3'-UTR元件(SEQ ID NO:1-16)的人工核酸分子的相对表达量大于1.9，具有更高的翻译效率。
表2.细胞表达验证结果
Figure PCTCN2022131289-appb-000144
Figure PCTCN2022131289-appb-000145
实施例5.LPP制剂的制备
5.1.实验材料
阳离子脂质SW-II-140-2为斯微生物合成；辅助磷脂(DOPE)采购自CordenPharma；胆固醇采购于Sigma-Aldrich；mPEG2000-DMG(即DMG-PEG 2000)采购于Avanti Polar Lipids,Inc.；PBS采购于Invitrogen；硫酸鱼精蛋白采购自北京斯利安药业有限公司。
5.2.mRNA的脂质多聚复合物(LPP)的制备
核酸水溶液的配制：用10mM柠檬酸-柠檬酸钠缓冲液(pH 4.0)将如实施例3制备的每种mRNA稀释为0.2mg/mL的mRNA溶液。
脂质溶液的配制：按照摩尔比例将SW-II-140-2：DOPE：胆固醇：mPEG2000-DMG以40:15:43.5:1.5溶解于乙醇溶液，配制成10mg/mL的脂质溶液。
硫酸鱼精蛋白溶液的配制：将硫酸鱼精蛋白溶解于无核酸酶水中配制成工作浓度为 0.25mg/mL的硫酸鱼精蛋白溶液。
核纳米颗粒(core nanoparticle)溶液的制备：使用微流控技术(迈安纳(上海)科技股份有限公司，型号：Inano D)，在以下条件将硫酸鱼精蛋白溶液与核酸溶液混合获得由鱼精蛋白和人工核酸分子形成的核纳米颗粒溶液：体积＝4.0mL；流速比＝5(mRNA):1(鱼精蛋白溶液)，总流速＝12mL/min，前废(start waste)＝0.35mL，后废(end waste)＝0.1mL，室温。
LPP的制备：在以下条件下将核纳米颗粒溶液与脂质溶液进行二次混合：体积＝4.0mL，流速比＝3(脂质溶液):1(核纳米颗粒溶液)，总流速＝12mL/min，前废＝0.35mL，后废＝0.1mL，室温，获得LPP溶液。
离心超滤：将LPP溶液通过超滤离心去除乙醇(离心力3000g，离心时间60min，温度4℃)，获得0.1mg/ml的包含不同人工核酸分子的LPP制剂。
本领域技术人员会清楚，可以进行本发明的许多修改和变化而不背离其精神和范围。本文所述的具体实施方案仅通过实例的方式提供，并不意味着以任何方式限制。本发明的真正范围和精神通过所附权利要求书示出，说明书和实施例仅是示例性的。
序列表
Figure PCTCN2022131289-appb-000146
Figure PCTCN2022131289-appb-000147
Figure PCTCN2022131289-appb-000148
Figure PCTCN2022131289-appb-000149
Figure PCTCN2022131289-appb-000150
Figure PCTCN2022131289-appb-000151
Figure PCTCN2022131289-appb-000152
Figure PCTCN2022131289-appb-000153
Figure PCTCN2022131289-appb-000154
Figure PCTCN2022131289-appb-000155
Figure PCTCN2022131289-appb-000156
Figure PCTCN2022131289-appb-000157

Claims (55)

  1. 一种人工核酸分子，其包含
    a.至少一个开放阅读框(ORF)；和
    b.至少一个3'-非翻译区元件(3'-UTR元件)，所述3'-UTR元件包含选自以下的核酸序列：
    (i)其中所述3'-UTR元件包含SEQ ID NO:44所示核酸序列的变体，所述变体与SEQ ID NO:44所示核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
    (ii)其中所述3'-UTR元件包含源自以下基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2、CVB3、AES和AAT，所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失；或者
    (iii)其中所述3'-UTR元件包含SEQ ID NO:16的核酸序列；
    或者上述核酸序列的相应RNA序列。
  2. 权利要求1的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:3的核酸序列。
  3. 权利要求2的人工核酸分子，其中所述3'-UTR元件还包含SEQ ID NO:92的核酸序列。
  4. 权利要求3的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:4的核酸序列。
  5. 权利要求1的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:44的核酸序列，并且还包含SEQ ID NO:90、91或93的核酸序列。
  6. 权利要求5的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:1、2或5的核酸序列。
  7. 权利要求1的人工核酸分子，其中所述核酸序列源自以下病毒基因的转录本的3'-UTR的核酸序列或其变体：HCV、CoV2和CVB3；或源自小鼠基因AES的转录本的3'-UTR的核酸序列或其变体；或源自人基因AAT的转录本的3'-UTR的核酸序列或其变体；或源自牛基因AES的转录本的3'-UTR的核酸序列或其变体，其中所述变体与所源自的核酸序列相比，其包含截短、末端延长和/或1、2、3个或更多个突变、添加或缺失。
  8. 权利要求7的人工核酸分子，其中所述3'-UTR元件呈现3-500个核苷酸、优选5-250个核苷酸、更优选90-215个核苷酸的长度。
  9. 权利要求8的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:6、7、8、9或12的核酸序列。
  10. 权利要求9的人工核酸分子，其中所述3'-UTR元件还包含SEQ ID NO:92的核酸序列。
  11. 权利要求10的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:10、14或15的核酸序列。
  12. 权利要求9的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:12的核酸序列，并且还包含SEQ ID NO:9或94的核酸序列。
  13. 权利要求12的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:11或13的核酸序列。
  14. 权利要求1-13中任一项的人工核酸分子，其还包含至少一个5'-非翻译区元件(5'-UTR元件)。
  15. 权利要求14的人工核酸分子，其中5'-UTR元件包含SEQ ID NO:45的核酸序列。
  16. 权利要求1-15中任一项的人工核酸分子，其还包含5'帽结构、聚胞苷酸序列、聚腺苷酸序列和/或组蛋白茎环；优选地，所述人工核酸分子包含聚腺苷酸序列；优选地，所述聚腺苷酸序列包含SEQ ID NO:46的核酸序列。
  17. 权利要求1-16中任一项的人工核酸分子，其中所述ORF是经过密码子优化的。
  18. 权利要求1-17中任一项的人工核酸分子，其是RNA，优选mRNA。
  19. 权利要求18的人工核酸分子，其中所述3'-UTR元件包含SEQ ID NO:47、48、49、50、51、52、53、54、55、56、57、58、59、60、61或62的核酸序列。
  20. 一种载体，其包含权利要求1-19中任一项的人工核酸分子。
  21. 权利要求20的载体，其是DNA载体。
  22. 权利要求20或21的载体，其是质粒载体或病毒载体，优选是质粒载体。
  23. 权利要求20-22中任一项的载体，其是环形分子。
  24. 一种细胞，其包含权利要求1-19中任一项的人工核酸分子或权利要求20-23中任一项的载体。
  25. 权利要求24的细胞，其是哺乳动物细胞；优选人受试者分离的细胞。
  26. 一种脂质组合物，其包含权利要求1-19中任一项的人工核酸分子以及包封所述人工核酸分子的脂质，其中所述包封人工核酸分子的脂质包含阳离子脂质、磷脂、类固醇和聚乙二醇修饰的脂质；所述脂质组合物还包含阳离子聚合物，其中所述阳离子聚合物与所述人工核酸分子缔合为复合物，共同包封在所述脂质中形成脂质多聚复合物。
  27. 权利要求26的脂质组合物，其中所述阳离子脂质包含式(I)的化合物，或其药学上可接受的盐
    Figure PCTCN2022131289-appb-100001
    其中，
    R 1和R 2各自独立选自键、C 1-C 12烷基和C 2-C 12烯基；
    R 3和R 4各自独立选自C 1-C 12烷基、C 2-C 12烯基、C 6-C 10芳基和5-10元杂芳基；并且R 3和R 4各自独立任选被t个R 6取代，t为选自1-5的整数；
    R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基；
    M 1和M 2各自独立地选自键、H、-O-、-S-、-C(O)-、-OC(O)-、-C(O)O-、-OC(O)O-、-SC(S)-、-C(S)S-、3-10元杂环、-NR 7-，或者
    R 5与M 1和M 2之一连同所连接的N原子一起形成3-10元杂环，且对应的R 1/R 3或者R 2/R 4不存在，所述杂环任选地被R 7取代；
    R 5选自C 3-8碳环、-C 1-12亚烷基-Q，Q选自H、-OR 7、-SR 7、-OC(O)R 7、-C(O)OR 7、 -N(R 7)C(O)R 7、-N(R 7)S(O) 2R 7、-N(R 7)C(S)R 7、-N(R 7) 2、氰基、C 3-8碳环、3-10元杂环、C 6-C 10芳基，上述基团各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代；
    m和n各自独立为选自0-12的整数；
    所述烷基、烯基和亚烷基各自任选地独立地被一个或多个选自以下的基团中断：-O-、-S-、-NR 7-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C 3-8碳环，且所述烷基、烯基和亚烷基各自任选地被一个或多个R 7取代；
    R 7各自独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、C 3-8碳环，上述基团各自任选地被一个或多个C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、C 6-C 10芳基、5-10元杂芳基、3-10元杂环、卤素、羟基、氧代(＝O)取代。
  28. 权利要求27的脂质组合物，其中，
    R 1和R 2各自独立选自C 1-C 12烷基和C 2-C 12烯基；
    其中R 3和R 4各自独立选自C 1-C 12烷基和C 2-C 12烯基；并且R 3和R 4各自独立任选被t个R 6取代，t为选自1-5的整数；R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基。
    M 1和M 2各自独立选自-OC(O)-、-C(O)O-、-OC(O)O-、-SC(S)-和-C(S)S-；
    R 5选自-C 1-12亚烷基-Q，Q选自-OR 7和-SR 7，R 7独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基和5-10元杂芳基；
    m和n各自独立为选自1-12的整数。
  29. 权利要求27的脂质组合物，其中所述阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐：
    Figure PCTCN2022131289-appb-100002
    Figure PCTCN2022131289-appb-100003
  30. 权利要求27的脂质组合物，其中所述阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐：
    Figure PCTCN2022131289-appb-100004
    Figure PCTCN2022131289-appb-100005
  31. 权利要求27的脂质组合物，其中所述阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐
    Figure PCTCN2022131289-appb-100006
    Figure PCTCN2022131289-appb-100007
  32. 权利要求27的脂质组合物，其中，
    R 1和R 2各自独立选自C 1-C 12烷基和C 2-C 12烯基；
    R 3和R 4各自独立选自C 1-C 12烷基、C 2-C 12烯基、C 6-C 10芳基和5-10元杂芳基；
    条件是R 3和R 4中至少一个为C 6-C 10芳基或5-10元杂芳基，并且R 3和R 4各自独立任选被t个R 6取代，t为选自1-5的整数；R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基；
    M 1和M 2各自独立选自-OC(O)-、-C(O)O-、-OC(O)O-、-SC(S)-和-C(S)S-；
    R 5选自-C 1-12亚烷基-Q，Q选自-OR 7和-SR 7，R 7独立选自H、C 1-C 12烷基、C 2-C 12烯基、C 1-C 12烷氧基、羧酸、亚磺酸、磺酸、磺酰基、硝基、氰基、氨基、氨甲酰基、磺酰胺、C 6-C 10芳基和5-10元杂芳基；
    m和n各自独立为选自1-12的整数。
  33. 权利要求32的脂质组合物，其中，
    R 1和R 2各自独立选自C 1-C 12烷基。
  34. 权利要求32或33的脂质组合物，其中，
    R 3和R 4各自独立选自C 1-C 12烷基和C 6-C 10芳基；
    条件是R 3和R 4之一为C 6-C 10芳基，另一个为C 1-C 12烷基；
    R 3和R 4各自独立被t个R 6取代，t为选自1-3的整数；
    R 6各自独立选自C 1-C 12烷基。
  35. 权利要求32-34中任一项所述的脂质组合物，其中，
    M 1和M 2各自独立选自：-OC(O)-、-C(O)O-和-OC(O)O-。
  36. 权利要求32-35中任一项所述的脂质组合物，其中，
    R 5选自-C 1-5亚烷基-Q，Q为-OH。
  37. 权利要求32-36中任一项所述的脂质组合物，其中，
    m和n各自独立为选自2-7的整数。
  38. 权利要求32-37中任一项所述的脂质组合物，其中，
    R 4取代于R 2的1位或末位；和/或
    R 3取代于R 1的1位或末位。
  39. 权利要求32-38中任一项所述的脂质组合物，其中，
    t为1或2，R 6取代于苯环上相对于R 1或R 2的间位和/或对位。
  40. 权利要求32-39中任一项所述的脂质组合物，其中，
    t为1或2，R 6各自独立选自C 1-C 10烷基。
  41. 权利要求32-40中任一项的脂质组合物，其中所述阳离子脂质包含式(II)的化合物，或其药学上可接受的盐：
    Figure PCTCN2022131289-appb-100008
    其中R 1、R 2、R 4、R 5、R 6、M 1、M 2、t、m和n如权利要求32-40中任一项所定义；
    优选地，在式(II)中
    R 1选自C 1-C 6烷基；
    R 2选自C 1-C 10烷基；
    R 4选自C 1-C 10烷基；
    M 1和M 2各自独立选自：-OC(O)-、-C(O)O-和-OC(O)O-；
    R 5选自-C 1-5亚烷基-Q，Q选自-OR 7和-SR 7，R 7独立选自H、C 1-C 12烷基和C 2-C 12烯基；
    m和n各自独立为选自2-9的整数；
    t为选自1-3的整数；
    R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基。
  42. 权利要求32-40中任一项的脂质组合物，其中所述阳离子脂质包含式(III)的化合物，或其药学上可接受的盐：
    Figure PCTCN2022131289-appb-100009
    其中R 1、R 2、R 4、R 5、R 6、t、m和n如权利要求32-40中任一项所定义；
    优选地，在式(III)中，
    R 1选自C 1-C 6烷基；
    R 2选自C 1-C 10烷基；
    R 4选自C 1-C 10烷基；
    R 5选自-C 1-3亚烷基-Q，Q选自-OH和-SH；
    t为1或2；
    R 6选自C 1-C 12烷基和C 2-C 12烯基；
    m和n各自独立为选自2-7的整数。
  43. 权利要求32-40中任一项的脂质组合物，其中所述阳离子脂质包含式(IV)的化合物，或其药学上可接受的盐：
    Figure PCTCN2022131289-appb-100010
    其中R 1、R 2、R 4、R 6、t、m和n如权利要求32-40中任一项所定义；
    优选地，在式(IV)中，
    R 1选自C 1-C 6烷基；
    R 2选自C 1-C 10烷基；
    R 4选自C 1-C 10烷基；
    t为1或2；
    R 6各自独立选自C 1-C 12烷基和C 2-C 12烯基；
    m和n各自独立为选自2-7的整数。
  44. 权利要求32的脂质组合物，其中所述阳离子脂质包含具有如下所示结构的脂质化合物或其药学上可接受的盐：
    Figure PCTCN2022131289-appb-100011
    Figure PCTCN2022131289-appb-100012
    优选地，所述阳离子脂质为SW-II-115、SW-II-121、SW-II-122、SW-II-134-3、SW-II-138-2、SW-II-139-2或SW-II-140-2。
  45. 权利要求27-44中任一项的脂质组合物，其中所述磷脂包含1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-双十一烷酰基-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0 Diether PC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16 Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME 16.0 PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺(LPE)或其组合；
    优选DSPC、DOPE或其组合。
  46. 权利要求45的脂质组合物，其中所述类固醇包含胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚及其衍生物；优选地，所述类固醇为胆固醇。
  47. 权利要求46的脂质组合物，其中所述聚乙二醇修饰的脂质包含1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)、1,2-二油酰基-rac-甘油,甲氧基-聚乙二醇(DOGPEG)和1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-聚(乙二醇)(DSPE-PEG)；优选DSPE-PEG、DMG-PEG或其组合。
  48. 权利要求47的脂质组合物，其包含
    10-70摩尔％的阳离子脂质、10-70摩尔％的磷脂、10-70摩尔％的类固醇和0.05-20摩尔％的聚乙二醇修饰的脂质；
    优选包含35-50摩尔％的阳离子脂质、10-30摩尔％的磷脂、24-44摩尔％的类固醇和1-1.5摩尔％的聚乙二醇修饰的脂质；和/或
    阳离子脂质、DOPE、胆固醇和DMG-PEG；
    优选包含50摩尔％的阳离子脂质、10摩尔％的DOPE、38.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG或40摩尔％的阳离子脂质、15摩尔％的DOPE、43.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG。
  49. 权利要求48的脂质组合物，其中所述阳离子聚合物包含聚-L-赖氨酸、鱼精蛋白、聚乙烯亚胺(PEI)或其组合；优选地，所述阳离子聚合物为鱼精蛋白。
  50. 一种药物组合物，其包含权利要求1-19中任一项的人工核酸分子、权利要求20-23中任一项的载体、权利要求24或25的细胞或权利要求26-49中任一项的脂质组合物，以及药学上可接受的载剂。
  51. 权利要求1-19中任一项的人工核酸分子、权利要求20-23中任一项的载体、权利要求24或25的细胞或权利要求26-49中任一项的脂质组合物或权利要求50的药物组合物在制备疫苗或用于基因治疗的药物中的用途。
  52. 权利要求1-19中任一项的人工核酸分子、权利要求20-23中任一项的载体、权利要求24或25的细胞或权利要求26-49中任一项的脂质组合物或权利要求50的药物组合物在制备用于治疗或预防疾病的药物中的用途。
  53. 一种用于增加人工核酸分子、优选mRNA分子或载体的翻译效率的方法，所述 方法包括连接开放阅读框与权利要求1-19中任一项所定义的3'-UTR元件。
  54. 一种试剂盒，其包含权利要求1-19中任一项的人工核酸分子、权利要求20-23中任一项的载体、权利要求24或25的细胞或权利要求26-49中任一项的脂质组合物或权利要求50的药物组合物。
  55. 一种产生人工核酸分子的方法，所述方法包括：
    a)合成权利要求1-19中任一项的人工核酸分子；或者
    b)通过20-23中任一项的载体合成人工核酸分子。
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