WO2023165530A1 - 提高rna分子的胞内翻译效率和/或稳定性的方法 - Google Patents

提高rna分子的胞内翻译效率和/或稳定性的方法 Download PDF

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WO2023165530A1
WO2023165530A1 PCT/CN2023/079088 CN2023079088W WO2023165530A1 WO 2023165530 A1 WO2023165530 A1 WO 2023165530A1 CN 2023079088 W CN2023079088 W CN 2023079088W WO 2023165530 A1 WO2023165530 A1 WO 2023165530A1
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modified
adenosine
poly
modification
sequence
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吴立刚
黎硕珂
张宏道
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中国科学院分子细胞科学卓越创新中心
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the present invention relates to methods for increasing the efficiency and stability of intracellular translation of RNA molecules.
  • the mRNA synthesized in vitro is delivered into cells by electroporation, liposome and other delivery technologies, and can be used to translate and express any natural or non-natural protein products at the cellular level and in animals, including microbial polypeptide antigens and tumor antigen polypeptide concatenations , antibodies, chimeric antigen receptors, natural and mutated forms of enzymes and cytokines, etc.
  • the method of using in vitro synthesized mRNA to express proteins in cells has been widely used in the development of various infectious disease vaccines, such as the approved new crown vaccine, the influenza virus vaccine that has entered phase III clinical trials, and the respiratory syncytial virus vaccine. and cytomegalovirus vaccines.
  • mRNA technology has also emerged in the fields of tumor immunotherapy and rare disease treatment. Currently, several mRNA drugs have entered phase I or phase II clinical trials.
  • nucleotide modifications that have been applied to mRNA technology such as pseudouridine ( ⁇ ) and 5-methylcytosine (m5C) modifications, are all aimed at mRNAs other than poly(A) Region or whole mRNA [Pardi, N., Hogan, MJ, Porter, FW, & Weissman, D. (2016). mRNA vaccines-a new era in vaccination. Nature reviews. Drug discovery, 17(4), 261-279 ].
  • poly(A) plays a key role in the translation efficiency and stability of mRNA [Passmore, LA, & Coller, J.
  • nucleotide modification in addition to base modification such as pseudouridine ( ⁇ ) modification, there are also modifications for phosphate groups and sugar groups, such as phosphorothioate (phosphorothioate, PS) modification, borophosphoric acid ( Phosphate group modification represented by boranophosphate (PB) modification, methylphosphonate (MP) modification and locked nucleic acid (LNA) modification, 2'-O-Methyl, 2' OMe) modification and 2'-fluoro (2'-Fluoro, 2'F) modification are representative glycosyl modifications.
  • phosphorothioate phosphorothioate
  • borophosphoric acid Phosphate group modification represented by boranophosphate (PB) modification
  • MP methylphosphonate
  • LNA locked nucleic acid
  • 2'-O-Methyl, 2' OMe modification and 2'-fluoro (2'-Fluoro, 2'F) modification are representative glycosyl modifications.
  • RNA modifications can greatly improve the stability of RNA, and have been successfully applied to a variety of antisense oligonucleotides (antisense oligonucleotides, ASO) drugs and small interfering RNA (small interfering RNA, siRNA) drugs [Duffy, K ., Arangundy-Franklin, S., & Holliger, P. (2020). Modified nuclear acids: replication, evolution, and next-generation therapeutics. BMC biology, 18(1), 112]. Whether these nucleotide modifications can be used to synthesize mRNA and improve the protein expression efficiency of mRNA has not been fully studied.
  • the first aspect of the present invention provides a method for preparing an RNA molecule, the method comprising adding modified adenylic acid and/or modified cytidylic acid and optionally Steps for modifying poly(A) sequences composed of unmodified adenine nucleotides.
  • the methods are used to prepare RNA molecules with improved translation efficiency and/or stability.
  • the poly(A) tail is 30-250, preferably 50-200, more preferably 60-200, more preferably 60-150 consecutive unmodified adenosine-containing Acid and optionally containing modified adenylic acid sequence 1
  • the modified poly (A) sequence is 1-250 in length, preferably 10-250, more preferably 20-200, more preferably 30-200 adenylate sequence 2 of nucleotides; preferably, at least 1%, preferably at least 5%, preferably 10-100%, more preferably 20-100%, more preferably 50-100% of said adenylate sequence 2
  • the nucleotides are modified adenine and/or modified cytidine.
  • the modified poly(A) sequence is added at the 3' end of the poly(A) tail of the starting RNA molecule using a ligation reaction or a polymerization reaction.
  • RNA ligase is used to connect the modified poly(A) sequence to the poly(A) tail; preferably, the modified poly(A) sequence
  • the 5' end has a 5'P modification and the 3' end has a modification selected from: 3'ddC, 3'BHQ-1, 3'BHQ-2, 3'MGB, 3'Dabcul, 3'6-TAMRA, 3' '6-FAM, 3'CY5, 3'CY3, 3'6-ROX, 3'inverted dT, 3'Biotin, 3'Biotin-TEG, 3'Amino modified C7, 3'Phosphate, 3'Digoxigenin, 3' Thiol modifier C6 S-S, 3'Thiol modifier C3 S-S, 3'Spacer C3, 3'Spacer C6, 3'Spacer 9, 3'Spacer 18, 3'dSpacer, 3'Cholesteryl-TEG, and 3'Ferrocene
  • a polymerase or terminal transferase is used to modify ATP and optionally unmodified ATP as a substrate, and at the poly(A) tail of the initial RNA molecule Add modified adenosine and optional unmodified adenosine at the 3' end; preferably, the reaction system of the polymerization reaction does not contain unmodified ATP, or when it contains unmodified ATP, the ratio of modified ATP to unmodified ATP is 1: 19 to 19:1 range, such as 1:9 to 9:1 or 2:8 to 8:2.
  • the starting RNA molecule contains modified nucleotides.
  • the method also includes the step of preparing a starting RNA molecule with the poly(A) tail by a DNA template; preferably, the DNA template contains from the 5' end to the 3' end A promoter sequence, a 5' untranslated region, a Kozak sequence, an open reading frame, a 3' untranslated region and a poly(A) tail sequence connected in sequence; more preferably, the DNA template is also followed by the poly(A) tail A ribozyme sequence is directly linked; preferably, the ribozyme is a ribozyme with self-cleaving function, preferably selected from HDV ribozyme, hairpin ribozyme and hammerhead ribozyme.
  • the modification in the modified adenylic acid and the modified cytidylic acid includes a modification for a phosphate group and a modification for a sugar group; preferably, the modification is selected from phosphorothioate (PS) Modification, phosphoroboronate (PB) modification, methylphosphonate (MP) modification, locked nucleic acid (LNA) modification, 2'methoxyl (2'OMe) modification and 2'fluoro (2'F) modification; preferred
  • the modified adenosine is PS-modified adenosine, PB-modified adenosine, MP-modified adenosine, LNA-modified adenosine, PS-modified adenosine and 2'OMe-modified adenosine
  • a second aspect of the present invention provides a method for preparing mRNA molecules, the method comprising the following steps:
  • step (1) (2) Adding modified adenylic acid and/or modified cytidylic acid and optional unmodified adenosine to the 3' end of the poly(A) tail of the mRNA molecule obtained in step (1) using a ligation reaction or a polymerization reaction acid or a modified poly(A) sequence containing said modified adenosine and/or modified cytidine and optionally unmodified adenine, thereby preparing said mRNA molecule.
  • the poly( A) The mRNA molecule is prepared by adding modified adenosine and optionally unmodified adenosine to the 3' end of the tail.
  • the DNA template in step (1), contains a promoter sequence, a 5' untranslated region, a Kozak sequence, an open reading frame, a 3' untranslated region and poly(A) tail; more preferably, the DNA template is also directly connected with a ribozyme sequence after the poly(A) tail; preferably, the ribozyme is a ribozyme with self-cleavage function , preferably selected from HDV ribozymes, hairpin ribozymes and hammerhead ribozymes.
  • the ratio of modified ATP to unmodified ATP is 1:19 to 19:1 range, such as 1:9 to 9:1 or 2:8 to 8:2.
  • the modified poly(A) sequence containing the modified adenosine and/or modified cytidine and optionally unmodified adenine is decomposed using RNA ligase It is connected to the 3' end of the poly(A) tail of the mRNA molecule; preferably, the 5' end of the modified poly(A) sequence has a 5'P modification, and the 3' end has a modification selected from the following: 3'ddC, 3'BHQ-1, 3'BHQ-2, 3'MGB, 3'Dabcul, 3'6-TAMRA, 3'6-FAM, 3'CY5, 3'CY3, 3'6-ROX, 3'inverted dT, 3'Biotin, 3'Biotin-TEG, 3'Amino modified C7, 3'Phosphate, 3'Digoxigenin, 3'Thiol modifier C6 SS, 3'Thiol modifier C3 SS, 3
  • the ratio of modified ATP to unmodified ATP is in the range of 1:19 to 19:1, Such as 1:9 to 9:1 or 2:8 to 8:2.
  • the third aspect of the present invention provides an RNA molecule having a poly(A ) tail; wherein, the poly(A) tail has a length of 30-250, preferably 50-200, more preferably 60-200, more preferably 60-150 consecutive nucleotides of the adenine sequence 1 and The 3' end of the adenine sequence 1 is connected to an adenine sequence 2 with a length of 1-250, preferably 10-250, more preferably 20-200, and more preferably 30-200 consecutive nucleotides; wherein , the adenine sequence 1 consists of unmodified adenine, or contains unmodified adenine and modified adenine, and the adenine sequence 2 contains at least one modified adenine and/or at least one Modified cytidine; Wherein, when the adenine sequence 1 contains modified adenine, thioadenosine is not the only modified adenine, preferably, the adenine sequence 1 does not contain thioadenos
  • the RNA molecule is selected from an mRNA molecule, a long non-coding RNA, or a small non-coding RNA precursor.
  • At least 1%, preferably at least 5%, preferably at least 10% of the nucleotides in the adenine sequence 2 are modified adenine and/or modified cytidine; preferably Preferably, 10-100%, more preferably 20-100%, more preferably 50-100% of the nucleotides in the adenine sequence 2 are modified adenine and/or modified cytidine.
  • the adenine sequence 1 consists of unmodified adenine
  • the adenine sequence 2 contains modified adenine and/or modified cytidine and optionally Unmodified adenosine
  • the first nucleotide connected between the adenine sequence 2 and the 3' end of the adenine sequence 1 is a modified adenine.
  • the modification in the modified adenylic acid and the modified cytidylic acid includes a modification for a phosphate group and a modification for a sugar group; preferably, the modification is selected from phosphorothioate (PS) Modification, phosphoroboronate (PB) modification, methylphosphonate (MP) modification, locked nucleic acid (LNA) modification, 2'methoxyl (2'OMe) modification and 2'fluoro (2'F) modification; preferred Ground, the modified adenosine is PS Modified adenosine, PB-modified adenosine, MP-modified adenosine, LNA-modified adenosine, mixture of PS-modified adenosine and 2'OMe-modified adenosine, PS-modified adenosine A mixture of acid and PB-modified adenylate, PS-modified adenine and MP-modified adenine
  • the mRNA molecule contains a 5' untranslated region, a Kozak sequence, an open reading frame, a 3' untranslated region and the poly(A) molecule connected in sequence from 5' to 3'.
  • the open reading frame encodes a protein or polypeptide molecule of interest; preferably, the protein or polypeptide molecule is selected from pathogen antigens, tumor antigens, cytokines, hormones, antibodies, chimeric antigens Receptors, enzymes and structural proteins.
  • the pathogen is selected from viruses, bacteria, fungi, spirochetes and parasites.
  • the tumor antigens are tumor-associated antigens and tumor-specific antigens.
  • the fourth aspect of the present invention provides a pharmaceutical composition, which contains the RNA molecule described in any embodiment herein or the RNA molecule or mRNA molecule prepared by the method described in any embodiment herein and pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a vaccine, preferably a prophylactic vaccine or a therapeutic vaccine, wherein the RNA molecule in the prophylactic vaccine encodes an antigenic polypeptide, and the RNA molecule in the therapeutic vaccine encodes therapeutic peptides.
  • a fifth aspect of the present invention provides an application selected from:
  • the poly(A) molecule is used in the RNA molecule of the existing poly(A) tail of preparation cell translation efficiency and/or stability improvement, or modified adenylic acid and/or modified cytidylic acid or modified adenosine
  • the modification is selected from the group consisting of phosphorothioate modification, phosphoroborate modification, phosphoromethyl modification, locked nucleic acid modification, 2' methoxy modification and 2' fluoro modification;
  • the ratio of modified ATP and/or modified CTP to unmodified ATP is 1:19 to 19:1 In the range, such as in the range of 1:9 to 9:1 or 2:8 to 8:2.
  • the poly(A) molecule consisting of modified adenosine and/or modified cytidine and optionally unmodified adenine does not contain unmodified adenine, or contains
  • the ratio of modified modified adenosine and/or modified cytidine to unmodified adenine is in the range of 1:19 to 19:1, such as 1:9 to 9:1 or 2:8 to the range of 8:2.
  • the sixth aspect of the present invention provides a kit, which contains a mixture of modified ATP and/or modified CTP and unmodified ATP, or contains modified adenylic acid and/or modified cytidylic acid and optionally A poly(A) molecule composed of unmodified adenine nucleotides, and the reagents needed to perform ligation or polymerization reactions.
  • the ratio of modified ATP and/or modified CTP to unmodified ATP is 1:19 to 19:1 within the range of 1:9 to 9:1 or 2:8 to 8:2; said modified adenylic acid and/or modified cytidylic acid and optionally unmodified adenylic acid
  • the poly(A) molecule does not contain unmodified adenosine, or when it does, the ratio of modified adenosine and/or modified cytidine to unmodified adenine is in the range of 1:19 to 19:1, as in In the range of 1:9 to 9:1 or 2:8 to 8:2.
  • the kit is used to implement the RNA preparation method or mRNA preparation method described in any embodiment herein.
  • Figure 1 Schematic representation of the poly(A) molecule of the invention.
  • the length of poly(A) is 31-500 adenine nucleotides, containing multiple unmodified adenine nucleotides and one or more modified adenine nucleotides; wherein, the 5' end of the poly(A) molecule is 30-250 A continuous unmodified adenylic acid sequence 1, the adenosic acid sequence 1 is followed by an adenosine sequence 2 with a modification ratio ⁇ 1% and a length of 1-250 nt.
  • Symbols "*" and "+” represent passing A nucleotide modification introduced by a ligation reaction or a polymerization reaction.
  • RNA oligo oligonucleotides (RNA) containing phosphorothioate (PS) modified adenosine and locked nucleic acid (LNA) modified adenosine to the 3' end of Fluc-A60 mRNA by ligation reaction oligonucleotides, abbreviated as RNA oligo) can improve the efficiency of mRNA protein expression in cells.
  • RNA oligo Structural diagram of unmodified adenosine, PS-modified adenosine, LNA-modified adenosine and LNA-modified cytidine incorporated into RNA molecules.
  • the symbol "*" represents PS modification, and the symbol "+” represents LNA modification.
  • the symbol “*" represents PS modification, and the symbol “+” represents LNA modification.
  • Figure 3 ATP (Adenosine-5′-O-(1-Thiotriphosphate), 1-Thio-ATP, ATP ⁇ S) modified by E.coli poly(A) polymerase and phosphorothioate (PS) in 70bp RNA fragment Add PS to modify adenine nucleotides at the 3' end.
  • PS-modified ATP ATP ⁇ S
  • ATP ⁇ S phosphorothioate
  • (c) ATP ⁇ S:ATP mixing ratios are 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0, 0:0, use E.coli poly(A) polymerase reaction to add poly(A) tails to 70bp RNA fragments, and the reaction products are detected by agarose gel electrophoresis.
  • FIG. 4 Using E.coli poly(A) polymerase and phosphorothioate (PS)-modified ATP (ATP ⁇ S) to add PS-modified adenosine at the 3' end of Fluc-A60 and Fluc-A0 mRNA can increase mRNA expression in cells expression efficiency within.
  • PS phosphorothioate
  • ATP ⁇ S phosphorothioate-modified ATP
  • FIG. 5 Using E.coli poly(A) polymerase, phosphorothioate (PS) modified ATP (ATP ⁇ S), 2' methoxy modified ATP (2'OMe-ATP), 2' fluoro modified ATP (2'F-dATP), borophosphate (PB) modified ATP (ATP ⁇ BH 3 ) and methylphosphonate (MP) modified ATP (ATP ⁇ CH 3 ) added modified adenosine at the 3' end of Fluc-A60 mRNA It can improve the expression efficiency of mRNA in cells.
  • PS phosphorothioate
  • ATP ⁇ S modified ATP
  • 2' methoxy modified ATP 2'OMe-ATP
  • 2'F-dATP 2' fluoro modified ATP
  • PB borophosphate
  • MP methylphosphonate
  • modified ATP ATP ⁇ CH 3
  • the symbol “#” represents 2' methoxy modification or 2' fluoro modification.
  • the symbol “*” represents PS modification, PB modification or MP modification.
  • the symbol “*” represents PS modification, PB modification or MP modification, and the symbol “#” represents 2'methoxy modification or 2'fluoro modification.
  • mRNA is tailed with ATP, 50% ATP+50% ATP ⁇ S, 50% ATP+25% ATP ⁇ S+25% 2'OMe-ATP, 50% ATP+25% ATP ⁇ S+25% 2'F-dATP To compare the protein expression efficiency of mRNA products of the same quality in cells.
  • (d) mRNA uses ATP, 50% ATP+50% ATP ⁇ S, 50% ATP+50% ATP ⁇ BH 3 , 50% ATP+50% ATP ⁇ CH 3 , 50% ATP+25% ATP ⁇ S+25% ATP ⁇ BH 3 , 50% ATP +25% ATP ⁇ S+25% ATP ⁇ CH 3 for tailing reaction to compare the protein expression efficiency of mRNA products of the same quality in cells.
  • FIG. 6 Using E.coli poly(A) polymerase and phosphorothioate (PS) modified ATP (ATP ⁇ S) in pseudouridine modification, 2'methoxyadenosine modification or 2'fluoroadenosine modification Adding PS-modified adenosine to the 3' end of Fluc-A60 mRNA can improve the protein expression efficiency of mRNA in cells.
  • PS phosphorothioate
  • ATP ⁇ S phosphorothioate
  • Figure 7 In vitro transcription template and HDV ribozyme structure of the present invention.
  • At the 3' end of the poly(A) sequence of the plasmid vector is the HDV ribozyme sequence.
  • This plasmid vector uses this plasmid vector to prepare a linearized DNA template for in vitro transcription reaction.
  • the 3' end ribozyme of the mRNA obtained is self-cleaved to form a uniform length , A uniform poly(A) tail with no non-A base residues.
  • Figure 8 Three schemes were used to construct plasmid vectors, prepare in vitro transcription templates, and synthesize mRNA.
  • (a) Use the Fluc-A100-RZ plasmid to prepare a DNA template, and synthesize RZ mRNA by in vitro transcription.
  • (b) Use Fluc-A100-NotIA-RZ plasmid to prepare DNA template, and synthesize NotIA-RZ mRNA by in vitro transcription.
  • c Using the Fluc-A100-NotIA-RZ plasmid, the restriction site NotI was used to prepare a DNA template, and the NotIcut mRNA was synthesized by in vitro transcription.
  • Figure 9 Detection of electrophoresis bands and protein expression efficiency of different mRNAs.
  • RZ, NotIA-RZ, and NotIcut before purification were electrophoresed with 6% PAGE denaturing gel, and ribozyme self-cut bands (marked by black triangles) could be observed.
  • b-d Protein expression efficiencies of RZ, NotIA-RZ, and NotIcut in HEK293T, HCT116, and Hela cells, respectively.
  • Poly(A) is an important structure at the 3' end of the mRNA molecule. Studies have shown that in cells, the poly(A) tail of mRNA binds to the poly(A) binding protein PABP, and PABP interacts with the EIF4G complex, which is crucial for the stability and translation of mRNA.
  • the present invention finds that after the 3' end of the existing poly(A) tail at the 3' end of the starting RNA molecule, a modified adenosine, a modified cytidine, or a modified adenine and a modified cytidine are introduced to form Extended poly(A) sequence, the resulting RNA molecule has Significantly improved translation efficiency and/or stability.
  • the present invention provides a method for preparing RNA molecules.
  • the RNA molecule prepared by the method has improved translation efficiency and/or stability in cells.
  • the method comprises the step of adding a modified poly(A) sequence comprising a modified adenine and/or a modified cytidine to the 3' end of an existing poly(A) tail of the starting RNA molecule.
  • the RNA molecule thus prepared has an extended poly(A) tail relative to the starting RNA molecule, and its extended part contains modified adenylic acid and/or modified cytidylic acid, which is more active than the starting RNA molecule or RNA molecules in which the poly(A) tail extension does not contain modified adenine and/or modified cytidine have improved intracellular translation efficiency and/or stability.
  • RNA stability refers to the degree of resistance of RNA molecules to degradation. RNA with high stability is not easy to degrade and has a longer half-life.
  • modified adenosine monophosphate (AMP) or modified cytidylic acid (CMP) refers to a modified adenosine monophosphate or cytidine monophosphate incorporated into an RNA molecule by chemical synthesis or using a polymerization method .
  • the modification in the modified adenylic acid and the modified cytidylic acid includes a modification to a phosphate group and a modification to a sugar group.
  • Modifications for phosphate groups include, but are not limited to, phosphorothioate (phosphorothioate, PS) modification, boranophosphate (PB) modification and methylphosphonate (methylphosphonate, MP) modification.
  • Modifications to sugar groups include but are not limited to locked nucleic acid (LNA) modification, 2'-methoxy (2'-O-Methyl, 2'OMe) modification and 2'-fluoro (2'-Fluoro, 2 'F) modification.
  • the modified adenosine in the extended poly(A) tail includes at least one of PS-modified adenosine, LNA-modified adenosine and LNA-modified cytidine .
  • RNA molecules with improved intracellular translation efficiency and/or stability of tails (may also be referred to as "RNA molecules with extended poly(A) tails").
  • the starting RNA molecule can be any RNA molecule known in the art, including but not limited to mRNA molecule, long non-coding RNA (lncRNA) or non-coding small RNA precursor (such as miRNA or siRNA precursors).
  • the starting RNA molecule can be an RNA molecule of any use of interest, including, but not limited to, diagnostic, prophylactic, and therapeutic uses of disease.
  • the starting RNA molecule can be an mRNA molecule containing sequentially linked 5' untranslated region, Kozak sequence, open reading frame, 3' untranslated region and poly(A) tail from 5' to 3' .
  • the length of the poly(A) tail of the starting mRNA molecule may be 30-250 adenosine, such as 50-200 adenine, 60-200 adenine, or 60-150 adenine.
  • the 5' untranslated region refers to the region located upstream of the open reading frame that is not translated into protein.
  • Kozak sequence refers to a sequence at the 5' end of eukaryotic mRNA, which plays an important role in translation initiation.
  • the 3' untranslated region (3'UTR) refers to the region downstream of the open reading frame that is not translated into protein. 5'UTR and 3'UTR are used to regulate mRNA translation, half-life and subcellular localization.
  • the 5'UTR, Kozak sequence and 3'UTR commonly used for mRNA preparation are well known in the art to practice the present invention.
  • UTRs from highly expressed genes are the first choice for the synthesis of mRNA.
  • UTRs can be optimized according to cell type, such as by removing miRNA-binding sites and AU-rich regions in the 3′ UTR to minimize mRNA degradation.
  • protein or polypeptide molecules include, but are not limited to, pathogen antigens, tumor antigens, cytokines, hormones, antibodies, chimeric antigen receptors, enzymes, and structural proteins.
  • pathogens include, but are not limited to, viruses, bacteria, fungi, spirochetes, and parasites.
  • Pathogen antigens may be immunogenic polypeptides from these pathogens, including antigenic peptides known in the art for use in vaccines to elicit an immunogenic response.
  • the protein or polypeptide molecule is an antigenic polypeptide.
  • the antigenic polypeptide is derived from a pathogen.
  • pathogens refer to microorganisms capable of causing infectious diseases in humans, animals and plants.
  • the pathogens include, but are not limited to, viruses, bacteria, fungi, protozoa, and/or parasites.
  • the pathogens include, but are not limited to, the following viruses: SARS-CoV-2, adenovirus, herpes simplex, encephalitis virus, papillomavirus, varicella-zoster virus, human cytomegalovirus, human herpesvirus, human Papillomavirus, Poliovirus, Hepatitis B virus, Norwalk virus, Coxsackie virus, Hepatitis A virus, Poliovirus, Severe acute respiratory syndrome virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza virus, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, Hendra virus, Nipah virus, rabies virus, hepatitis D virus, round Virus
  • the protein or polypeptide molecule is a tumor antigen.
  • tumor-associated antigens tumor-associated antigens
  • tumor-specific antigens tumor-specific antigens, also known as tumor neoantigens
  • the tumor-associated antigen is an antigen molecule that exists on both normal cells and tumor cells, and is not unique to tumor cells. It is usually highly expressed when tumor cells proliferate.
  • Exemplary tumor-associated antigens include: embryonic antigens, glycoprotein antigens, squamous cell antigens, and the like. More specifically, tumor-associated antigens include, but are not limited to, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), New York esophageal squamous cell carcinoma antigen 1 (NY-ESO-1), melanoma-associated antigen A3 (MAGE- A3) and tyrosinase etc.
  • tumor neoantigens are produced by non-synonymous mutations in tumor cells, and are only expressed on the surface of tumor cells, and do not exist on any normal cells at different developmental stages.
  • cytokines, hormones, antibodies, enzymes and structural proteins described herein may be proteins or polypeptides known in the art with therapeutic or preventive activities, including natural proteins or functional peptides, and optimized mutant proteins.
  • the chimeric antigen receptor (CAR) described herein may be the currently known CAR for various antigens (especially tumor-associated antigens).
  • exemplary antigens include, but are not limited to, CD19, CD20, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, MSLN, CA125, PD1, PD-L1, FAP, and EGFR, among others.
  • the structure of CAR is well known in the art, and an exemplary CAR generally includes a signal peptide, an antibody sequence, a hinge region, a transmembrane region, and an intracellular signal transduction region.
  • the open reading frame is codon optimized.
  • modified nucleotides may be contained in the starting RNA molecule. Modified nuclei known in the art Nucleotides can be included in the starting RNA molecule. Likewise, modifications include modifications to phosphate groups and/or modifications to sugar groups. Modified nucleotides may be modified adenylic acid, modified guanylic acid, modified cytidylic acid and modified uridine acid. Modifications for phosphate groups include but are not limited to phosphorothioate (PS) modification, boranophosphate (PB) modification, methylphosphonate (MP) modification, mesyl-phosphoramidate , MsPA) modification.
  • PS phosphorothioate
  • PB boranophosphate
  • MP methylphosphonate
  • MsPA mesyl-phosphoramidate
  • Modifications on sugar groups include but are not limited to locked nucleic acid (LNA) modification, restricted ethyl bridged nucleic acid (cEt) modification, ethylene-bridged nucleic acid (ENA) modification ; 2'-methoxy (2'-O-methyl, 2'OMe) modification, 2'-fluoro (2'-fluoro, 2'F) modification, 2'-oxo-methoxyethyl (2'-O -methoxyethyl, 2'MOE) modification.
  • Modifications on bases include, but are not limited to, methylation, fluorine, thio, and bromine modifications.
  • Exemplary modifications in the starting RNA molecule include, but are not limited to, pseudouridine, 5-methyluridine, N1-methylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxy Uridine, 5-methylcytidine, 2'-fluorouridine, 2'-fluoroguanosine, 5-bromouridine, ⁇ -thiocytidine, N1-methyladenosine, 5- Methylcytidine and phosphorothioate-modified adenosine, locked nucleic acid-modified adenosine, 2'methoxyadenosine and 2'fluoroadenosine, etc.
  • nucleosides and nucleotides can be found in CN103974724A and Roberts, TC, etc., [2020, "Advances in oligonucleotide drug delivery. Nature reviews", Drug discovery, 19(10), 673-694], cited herein It is incorporated in its entirety into this article.
  • modified nucleotides need not be evenly distributed along the entire length of the starting RNA molecule.
  • Various nucleotide modifications may be present at various positions in the starting RNA molecule.
  • Modified nucleotides may be present anywhere in the starting RNA molecule including, for example, in the 5' untranslated region, Kozak sequence, open reading frame, 3' untranslated region and poly(A) of the starting mRNA molecule. Any one or more modified nucleotides are present in any one or more regions in the tail.
  • a modified poly(A) sequence containing modified adenine and/or modified cytidine can be added to the 3' end of the existing poly(A) tail of the starting RNA molecule by ligation reaction or polymerization reaction.
  • RNA oligonucleotides RNA oligonucleotides, abbreviated as RNA oligo
  • RNA oligo oligonucleotides
  • the poly(A) tail joins.
  • the oligonucleotide sequence contains one or more modified adenine and/or modified cytidine.
  • the oligonucleotide sequence consists of modified adenine and/or modified cytidine.
  • the oligonucleotide sequence consists of modified adenosine and/or modified cytidine and unmodified adenine.
  • the oligonucleotide sequence contains at least 1%, preferably at least 5%, preferably at least 10% of modified adenosine and/or modified cytidine.
  • the ratio of modified adenylic acid and/or modified cytidylic acid is 5-100%, such as 10-100%, 20-100%, 30-100%, 40- 100%, 50-100%, etc., or may be 5-60%, 10-60%, 15-60%, or 20-50% etc.
  • the oligonucleotide sequence is the adenine sequence 2 described in any embodiment herein.
  • the 5' end of the oligonucleotide sequence may have a 5'P modification, and the 3' end may have a modification selected from: 3'ddC, 3'BHQ-1, 3'BHQ-2 , 3'MGB, 3'Dabcul, 3'6-TAMRA, 3'6-FAM, 3'CY5, 3'CY3, 3'6-ROX, 3'inverted dT, 3'Biotin, 3'Biotin-TEG, 3'Amino modified C7, 3'Phosphate, 3'Digoxigenin, 3'Thiol modifier C6 S-S, 3'Thiol modifier C3 S-S, 3'Spacer C3, 3'Spacer C6, 3'Spacer 9, 3'Spacer 18, 3' dSpacer, 3'Cholesteryl-TEG, 3'Ferrocene dT.
  • 3'ddC 3'BHQ-1, 3'BHQ-2 , 3'MGB, 3'Dabcul
  • the reaction system of the ligation reaction may contain the initial RNA molecule, the oligonucleotide sequence, ATP, reaction buffer, ligase, RNase inhibitor and the like.
  • Specific reaction conditions may be conventional reaction conditions.
  • modified ATP and/or modified CTP can be used as substrates, and enzymes can be used to add unmodified poly(A) to the 3' end of the mRNA molecule.
  • Nucleotides and Modified Nucleotides are examples of nucleotides.
  • Enzymes that can be used in the present invention to add adenylic acid and/or cytidylic acid at the 3' end of RNA can be various polymerases and terminal transferases known in the art, and the polymerases include various enzymes that can be single-stranded RNA molecules 3 RNA and DNA polymerases that add ATP, UTP, CTP, and/or GTP to the 'end.
  • Exemplary polymerases include, but are not limited to, poly(A) polymerase (eg, E. coli poly(A) polymerase), poly(U) polymerase, and DNA polymerase theta (DNA polymerase theta, POLQ).
  • Exemplary terminal transferases include various RNA terminal transferases and terminal deoxynucleotidyl transferases (TdT).
  • the polymerization reaction can be carried out using one or more suitable enzymes as needed.
  • poly(A) polymerase such as E. coli poly(A) polymerase Synthases carry out the polymerization reactions described herein.
  • the reaction system of the polymerization reaction may contain starting RNA molecules, modified ATP and/or modified CTP, optional unmodified ATP, reaction buffer, RNase inhibitors, enzymes and other components.
  • the reaction can be carried out under conventional reaction conditions.
  • the reaction system does not contain unmodified ATP.
  • the reaction system contains modified ATP and/or modified CTP and unmodified ATP, wherein the ratio of modified ATP and/or modified CTP to unmodified ATP is 1:19 to 19:1 range, such as 1:9 to 9:1, 2:8 to 8:2 or 4:6 to 6:4.
  • the ratio of modified ATP and/or modified CTP to unmodified ATP is about 5:5.
  • starting RNA molecules can be prepared by methods well known in the art.
  • the preparation method of the starting mRNA molecule as the starting RNA molecule may include: using poly(A) polymerase to add tails; using DNA primers with poly(A) to perform PCR amplification to obtain poly(A)-containing DNA templates, poly(A)-containing RNAs are prepared by in vitro transcription (IVT); DNA templates with poly(A) can also be obtained by directly inserting poly(A) sequences into plasmid vectors.
  • Plasmid vectors are currently the most widely used mainstream method. Plasmid vectors are linearized before being used as templates for in vitro transcription, and restriction enzyme cleavage sites need to be introduced after the poly(A) sequence of the plasmid DNA template.
  • starting mRNA molecules are prepared by in vitro transcription using a DNA template.
  • the starting mRNA molecule is prepared using a DNA template with the following structure: the DNA template contains a promoter sequence, a 5' untranslated region, a Kozak sequence connected in sequence from the 5' end to the 3' end , open reading frame, 3' untranslated region, poly(A) tail and ribozyme sequence.
  • the 5' untranslated region, Kozak sequence, open reading frame and 3' untranslated region are as described in any of the preceding embodiments.
  • a ribozyme refers to an RNA molecule with catalytic activity, whose chemical essence is ribonucleic acid (RNA), but which has the catalytic function of an enzyme.
  • the ribozymes of the present invention are ribozymes with self-cleavage function, including but not limited to HDV ribozymes, hairpin ribozymes, and hammerhead ribozymes.
  • Other nucleotide sequences that can be cleaved autocatalytically or mediated by protein factors are also useful in the present invention.
  • the present invention uses the HDV ribozyme whose nucleotide sequence is shown in positions 113-197 of SEQ ID NO:1.
  • the promoter may be various promoters used in the art to prepare mRNA.
  • Promoters include those of eukaryotic expression systems and prokaryotic expression systems.
  • Exemplary promoters of eukaryotic expression systems include, but are not limited to, CMV promoters, EF1a promoters, SV40 promoters, PGK1 promoters, CAG promoters, TRE promoters, GDS promoters, Ac5 promoters, ADH1 promoters, Ubc promoter, UAS promoter, GAL1 promoter, TEF1 promoter, H1 promoter and U6 promoter, etc.
  • Commonly used prokaryotic expression system promoters include T7 promoter, T7lac promoter, Sp6 promoter, araBAD promoter, trp promoter, lac promoter, Ptac promoter and pL promoter, etc.
  • the DNA template can be provided in the form of a vector.
  • Exemplary vectors include, but are not limited to, pUC19, pBR322, pBluescript, and the like.
  • suitable enzyme cutting sites are provided at both ends of the DNA template.
  • the vectors of the present invention can be constructed using methods well known in the art.
  • the vectors of the invention are plasmid vectors capable of autonomous replication in a host cell.
  • T4 polynucleotide kinase can be used for treatment, followed by the next ligation reaction or polymerization reaction.
  • the length of the new full-length poly(A) tail obtained after the extension of the poly(A) tail in the RNA molecule can be 31-500 adenine nucleotides, which contains multiple unmodified adenosine acid and one or more modified adenosine.
  • the existing poly(A) tail of an RNA molecule can be regarded as an adenine nucleotide sequence 1, and its length is usually 30-250 consecutive adenine nucleotides.
  • the poly(A) tail extended outside the poly(A) tail Part A) is considered to be an adenine sequence 2, which is usually 1-250 nucleotides in length.
  • the adenine sequence 1 may consist of unmodified adenine, or contain unmodified adenine and modified adenine.
  • Adenine sequence 2 contains at least one modified adenine.
  • the length of the adenine sequence 1 is at least 50 nucleotides, preferably at least 60 nucleotides. In some embodiments, the length of the adenine sequence 1 is 50-200 nucleotides. In some embodiments, the adenine sequence is 60-200 nucleotides in length. In some embodiments, the length of the adenine sequence 1 is 60-150 nucleotides.
  • thioadenosine is not the only modified adenine. In some embodiments, the adenine sequence 1 does not contain thioadenosine modifications.
  • the adenine sequence 2 contains modified adenine and/or modified cytidine and Optional unmodified adenosine. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 10 nucleotides of the adenine sequence 2 are modified adenine and/or modified cytidine. In some embodiments, at least 1% of the nucleotides in the adenine sequence 2 are modified adenines and/or modified cytidines. In some embodiments, at least 5% of the nucleotides in the adenine sequence 2 are modified adenines and/or modified cytidines.
  • At least 10% of the nucleotides in the adenine sequence 2 are modified adenines and/or modified cytidines.
  • 1-100% of the nucleotides in the adenine sequence 2 are modified adenine and/or modified cytidine, such as 1-100%, 10-100%, 20-100% , 30-100%, 40-100%, 50-100%, etc., or may be 1-60%, 5-60%, 10-60%, 15-60%, or 20-50%, etc.
  • the modified adenosine at least includes PS-modified adenosine.
  • the modified adenosine acid is PS-modified adenosine acid .
  • all modified adenosines are PS-modified adenosines.
  • the modified cytidine contained in the adenine sequence 2 is LNA modified cytidine.
  • the adenine sequence 2 is more than 5 nucleotides in length. In some embodiments, the adenine sequence 2 is more than 10 nucleotides in length. In some embodiments, the adenine sequence 2 is 10-250 nucleotides in length. In some embodiments, the adenine sequence 2 is 20-200 nucleotides in length. In some embodiments, the adenine sequence 2 is 30-200 nucleotides in length. In some embodiments, the adenine sequence 2 is less than 150 nucleotides in length.
  • the length of the adenine sequence 2 is 5-150 nucleotides, 10-150 nucleotides, 20-150 nucleotides, 30-150 nucleotides, 20-100 nucleotides nucleotides, 20-50 nucleotides, etc.
  • the adenine sequence 1 consists of unmodified adenine
  • the adenine sequence 2 contains modified adenine and/or modified cytidine and optionally unmodified adenine nucleotides
  • the first nucleotide connected between the adenosine sequence 2 and the 3' end of the adenosine sequence 1 is a modified adenosine.
  • the starting RNA molecule has a 5' cap structure, and thus the prepared RNA molecule further comprises at least one 5' cap structure.
  • Exemplary 5' cap structures include, but are not limited to, Cap0 (m7GpppXpYp), Cap1 (m7GpppXmpYp), Cap2 (m7GpppXmpYmp); Anti-reverse cap analog (ARCA), ⁇ -S-ARCA, 5'-LNA-Cap, 1,5'-LNA-Cap and N7-benzyl dinucleoside tetraphosphate cap analog.
  • the RNA molecule can be capped using techniques well known in the art.
  • RNA molecule after the mRNA molecule is prepared, it is subjected to 5' capping and 3' adding poly(A) tail treatment to obtain the starting RNA molecule described herein, and then the method described herein is used to extend the poly(A) tail.
  • the present invention provides an RNA molecule.
  • the RNA molecule has improved intracellular translation efficiency and/or stability.
  • the RNA molecule has a poly(A) tail, and the length of the poly(A) tail is 31-500 adenine nucleotides, which contains a plurality of unmodified adenine nucleotides and one or more modified adenine nucleotides and/or One or more modified cytidines; the poly(A) tail has an adenine sequence 1 of 30-250 contiguous nucleotides in length and an adenine sequence 2 of 1-250 contiguous nucleotides in length .
  • the adenine sequence 1 may consist of unmodified adenine, or contain unmodified adenine and modified adenine.
  • the adenine sequence 2 contains at least one modified adenine and/or at least one modified cytidine. In some embodiments, the adenine sequences 1 and 2 are as described in any of the preceding embodiments.
  • thioadenosine is not the only modified adenine; in some embodiments, the adenine sequence 1 does not contain Thioadenosine modification; in some other embodiments, the adenylic acid sequence 1 is composed of unmodified adenosine acid, and the adenylic acid sequence 2 contains modified adenosine acid and/or modified cytidylic acid
  • the first nucleotide linked to the 3' end of the adenylic acid sequence 1 is a modified adenosic acid with an optional unmodified adenosine acid.
  • the adenine sequence 1 is 50-200, preferably 60-200, more preferably 60-150 nucleotides in length
  • the adenine sequence 2 is 1-250 in length, such as 5-200 1, or 10-200, or 10-150 nucleotides, and 1-100% of the nucleotides in the adenine sequence 2 are modified adenosine and/or modified cytidine, such as 5 -100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, etc., or may be 5-60%, 10-60%, 15-60% or 20- 50% etc.
  • the RNA molecule is an mRNA molecule.
  • the mRNA molecule has the biological function and structure described in any of the above embodiments.
  • the RNA molecule is prepared by the RNA molecule preparation method described in any embodiment herein.
  • the present application provides a poly(A) molecule.
  • the poly(A) molecule can exist independently or as part of an RNA molecule. When part of an RNA molecule, its presence contributes to increased translation efficiency and/or stability of the RNA molecule within the cell.
  • the length of the poly(A) molecule is 31-500 adenine acid, which contains a plurality of unmodified adenine acid and one or more modified adenine acid and/or one or more modified cytidine;
  • the poly(A) tail has an adenine sequence 1 of 30-250 consecutive nucleotides in length and an adenine sequence 2 of 1-250 consecutive nucleotides in length.
  • the adenine sequence 1 may consist of unmodified adenine, or contain unmodified adenine and modified adenine.
  • the adenine sequence 2 contains at least one modified adenine and/or at least one modified cytidine. In some embodiments, the adenine sequences 1 and 2 are as described in any of the preceding embodiments.
  • thioadenosine is not the only modified adenine; in some embodiments, the adenine sequence 1 does not contain Thioadenosine modification; in some other embodiments, the adenylic acid sequence 1 is composed of unmodified adenosine acid, and the adenylic acid sequence 2 contains modified adenosine acid and/or modified cytidylic acid
  • the first nucleotide linked to the 3' end of the adenylic acid sequence 1 is a modified adenosic acid with an optional unmodified adenosine acid.
  • the adenine sequence 1 is 50-200, preferably 60-200, more preferably 60-150 nucleotides in length
  • the adenine sequence 2 is 1-250 in length, such as 5-200 1, or 10-200, or 10-150 nucleotides, and 1-100% of the nucleotides in the adenine sequence 2 are modified adenosine and/or modified cytidine, such as 5 -100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, etc., or may be 5-60%, 10-60%, 15-60% or 20- 50% etc.
  • the present invention provides a pharmaceutical composition comprising the RNA molecule according to any embodiment of the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a vaccine, ie, a nucleic acid vaccine.
  • the vaccine is a tumor vaccine.
  • the vaccines are prophylactic and therapeutic vaccines.
  • the mRNA molecule in the preventive vaccine encodes the antigenic polypeptide.
  • mRNA molecules encode therapeutic polypeptides, including tumor-associated antigen polypeptides (TAA) and tumor-specific antigen polypeptides (TSA).
  • the pharmaceutically acceptable carrier may be a pharmaceutically acceptable carrier, excipient, etc. known in the art for delivering mRNA molecules.
  • the pharmaceutical composition is a vaccine comprising said mRNA molecule and an adjuvant.
  • the adjuvant can be a common adjuvant for mRNA vaccines.
  • the invention provides modified ATP, modified CTP, mixture of modified ATP and unmodified ATP, mixture of modified CTP and unmodified ATP, modified ATP and modified CTP and unmodified ATP described in any embodiment herein.
  • the starting RNA molecules used in said preparation already have a poly(A) tail.
  • the invention provides modified adenylic acid and/or modified cytidylic acid or a poly(A) molecule consisting of modified adenylic acid and/or modified cytidylic acid and optionally unmodified adenylic acid Use in improving intracellular translation efficiency and/or stability of RNA molecules having poly(A) tails.
  • the poly(A) tail is the adenine sequence 1 described in any embodiment of the present application.
  • the mixture of modified ATP and unmodified ATP in the mixture of modified ATP and unmodified ATP, the mixture of modified CTP and unmodified ATP, the mixture of modified ATP and modified CTP and unmodified ATP, modified ATP and/or modified CTP and unmodified ATP
  • the ratio of modified ATP is in the range of 1:19 to 19:1, such as in the range of 1:9 to 9:1, 2:8 to 8:2 or 4:6 to 6:4, or may be 5: 5.
  • the poly(A) molecule composed of modified adenosine and/or modified cytidine and optionally unmodified adenine is the adenine sequence 2 described in any embodiment herein .
  • the poly(A) molecule consisting of modified adenosine and/or modified cytidine and optionally unmodified adenosine contains no unmodified adenosine, or when it does, the modified adenosine
  • the ratio of nucleotide and/or modified cytidylic acid to optionally unmodified adenylic acid is in the range of 1:19 to 19:1, such as 1:9 to 9:1, 2:8 to 8:2 or In the range of 4:6 to 6:4, or may be 5:5.
  • the modifications include modifications to phosphate groups and modifications to sugar groups, including but not limited to phosphorothioate (phosphorothioate, PS) modification, boranophosphate (PB) modification, methyl substitution Phosphate group modification represented by methylphosphonate (MP) modification and locked nucleic acid (LNA) modification, 2'-methoxy (2'-O-Methyl, 2'OMe) modification, 2' fluoro( 2'-Fluoro, 2'F) modification is a representative glycosyl modification.
  • phosphorothioate phosphorothioate
  • PB boranophosphate
  • 2'-methoxy (2'-O-Methyl, 2'OMe) modification 2' fluoro( 2'-Fluoro, 2'F) modification is a representative glycosyl modification.
  • the modified ATP is PS-modified ATP, LNA-modified ATP, PB-modified ATP, MP-modified ATP, a mixture of PS-modified ATP and 2'OMe-modified ATP, PS-modified ATP and A mixture of 2'F modified ATP, a mixture of PS modified ATP and PB modified ATP, or a mixture of PS modified ATP and MP modified ATP.
  • modified CTPs include LNA-modified CTPs.
  • the present invention also provides a kit, which contains the mixture of modified ATP and/or modified CTP and unmodified ATP described in any embodiment herein, or contains the modified adenosine and/or modified ATP described in any embodiment herein Or a poly(A) molecule consisting of modified cytidine and optionally unmodified adenine.
  • the kits of the present invention can be used to add modified adenosine nucleotides to the 3' end of a poly(A) tail already possessed by an RNA molecule by polymerization or ligation, particularly as described in any of the embodiments herein. And/or a poly(A) sequence of modified cytidine and optionally unmodified adenine.
  • kits of the present invention may also contain reagents for carrying out polymerization or ligation reactions.
  • reagents for performing ligation include but are not limited to ATP/CTP, reaction buffer, and ligase, etc.
  • reagents for performing polymerization include enzymes (polymerase and/or terminal transferase as described above, poly(A) polymerase, especially E.coli poly(A) polymerase), RNase inhibitor and reaction buffer, etc.
  • the kit of the invention further comprises reagents for preparing the RNA molecule to be subjected to the ligation reaction or polymerization reaction.
  • Exemplary methods for preparing mRNA molecules include: using poly(A) polymerase to add tails; using poly(A) DNA primers to perform PCR amplification to obtain poly(A)-containing DNA templates, and in vitro transcription (IVT ) preparing poly(A)-containing RNA; and directly inserting the poly(A) sequence into a plasmid vector to obtain a poly(A)-containing DNA template, and preparing poly(A)-containing RNA by in vitro transcription (IVT).
  • Preparation of RNA molecules may or may not include 5' capping reactions. Accordingly, the kits of the invention may contain the reagents necessary to practice these methods/steps.
  • the present invention is different from the method of adding modified nucleotides in the whole mRNA or in the whole poly(A) tail, but adds a modified polynucleotide to the 3' end of the existing mRNA poly(A) tail. Nucleotides. This method of adding additional modified nucleotides only to the back of the poly(A) tail at the 3' end of the mRNA significantly improves the protein expression efficiency of the mRNA.
  • Plasmid vectors of firefly luciferase reporter gene (Fluc) and Renilla luciferase (Rluc) reporter gene were constructed, and DNA templates for PCR amplification were obtained through enzyme digestion and purification.
  • poly(A)-containing DNA primers for PCR amplification poly(A)-containing DNA fragments can be obtained as templates for in vitro transcription of RNA.
  • forward primer forward primer
  • reverse primer reverse primer
  • reaction conditions three-step method according to the instructions, after 3 minutes at 95°C, react for 30 cycles, and finally extend at 68°C for 5 minutes.
  • the cycle conditions were: denaturation at 98°C for 10 seconds, annealing at 56°C for 30 seconds, and extension at 68°C for 3 minutes.
  • the product was purified using a DNA purification kit.
  • Poly(A)-containing mRNA is prepared by in vitro transcription (IVT) using a poly(A)-containing DNA template.
  • IVT in vitro transcription
  • cap analogue Cap1 at a final concentration of 1.6mM and ATP, UTP, CTP, GTP at a final concentration of 2mM, as well as 20ng/ul DNA template, 1 ⁇ T7 reaction buffer, T7 RNA polymerase, RNase inhibitor, reacted at 37°C for 4 hours to stop the reaction, added DNase I and reacted at 37°C for 15 minutes to remove the DNA template, and then purified the product using the MEGAclear Transcription Purification Kit (Thermo Fisher).
  • the ATP/UTP/CTP/GTP part in the in vitro transcription reaction system can be replaced by modified ATP/UTP/CTP/GTP.
  • modify ATP/UTP/CTP/GTP replace part of UTP with pseudouridine-modified UTP, and replace part of ATP with 2'methoxyadenosine or 2'fluoroadenosine-modified ATP.
  • RNA ligase 1 was used to carry out the ligation reaction between mRNA and RNA oligonucleotides (referred to as RNA oligo).
  • RNA oligos are modified with 5'P and 3'ddC.
  • a 20ul ligation reaction use 2pmol mRNA and 50pmol RNA oligo, and 1mM ATP, 20% PEG8000, 10% DMSO, 1 ⁇ T4 ligase reaction buffer, T4 ligase I, RNase inhibitor, reacted at 25°C for 2 hours to stop the reaction, and the product was purified using MEGAclear transcription purification kit (Thermo Fisher). purification.
  • E.coli poly(A) polymerase Using E.coli poly(A) polymerase, ATP and modified ATP are used as substrates to add modified nucleotides to the 3' end of RNA.
  • ATP and modified ATP are used as substrates to add modified nucleotides to the 3' end of RNA.
  • E.coli poly(A) polymerase In a 20ul tailing reaction, use 10ug RNA and a final concentration of 1mM ATP and modified ATP, as well as 1 ⁇ E.coli poly(A) polymerase reaction buffer, E.coli poly(A) polymerase, RNase Inhibitors were reacted at 37°C for 15-60 minutes to terminate the reaction, and the product was purified using the MEGAclear Transcription Purification Kit (Thermo Fisher).
  • a 1.5% agarose gel was prepared, and the RNA obtained after the tailing reaction using E.coli poly(A) polymerase was electrophoresed at a voltage of 160V.
  • Fluc for short mRNAs Different firefly luciferase (Fluc for short) mRNAs were used to transfect the cells, and an equal amount of renilla luciferase (Rluc for short) reporter gene mRNA was simultaneously transfected as a reference.
  • Rluc for short reporter gene mRNA was simultaneously transfected as a reference.
  • HEK293T cell density is about 40%, dilute 0.22 ⁇ l lipofectamine2000 in 50 ⁇ l Opti-MEM, and after standing at room temperature for 5 minutes, add 100 ng Fluc mRNA and 10 ng diluted in 50 ⁇ l Opti-MEM Rluc mRNA, mix well and then stand at room temperature for 20 minutes, and finally carefully add to the cells, after 24, 36, and 48 hours of incubation, rinse the cells with 1 ⁇ PBS, and then use an appropriate amount of lysis buffer (100mM Potassium Phosphate [pH 7.8 ], 0.2% (v/v) Triton X-100) to lyse the cells, centrifuge at 10,000rpm at room temperature for 1 minute, take an appropriate amount of supernatant, and add the corresponding volume of the luciferase reaction base provided in the dual luciferase reporter gene detection kit Fluorescence values were recorded with a multifunctional microplate detector.
  • lysis buffer 100mM Potassium Phosphate
  • A20-PS RNA oligo containing phosphorothioate (PS) modification (containing only 3 phosphorothioate modified A), containing locked nucleic acid (locked nucleic acid acid, LNA) modified A20-LNA RNA oligo (only contains 2 locked nucleic acid modified A), contains locked nucleic acid (LNA) modified A20-LNAC RNA oligo (only contains 2 locked nucleic acid modified C) and does not contain Modified A20 RNA oligo (Fig. 2, a-b).
  • PS phosphorothioate
  • Phosphorothioate-modified adenosine triphosphate is also called ATP ⁇ S (Adenosine-5′-O-(1-Thiotriphosphate), 1-Thio-ATP), the structure of which is shown in Figure 3(a).
  • ATP ⁇ S:ATP mixing ratio we first set the total final concentration of ATP and ATP ⁇ S in the reaction system as 1 mM, and the set ATP ⁇ S:ATP ratios were 0:10, 1:9, 2:8, 3 :7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0, 0:0.
  • RNA obtained through the tailing reaction of 30 min and 60 min was electrophoresed on a 1.5% agarose gel, and the length of the tail was judged by the size change of the RNA band.
  • RNA bands did not shift significantly; when the ATP ⁇ S:ATP ratio was 0:10, 1:9, 2:8 , 3:7, 4:6, 5:5, and 6:4, the RNA migrated significantly, and when the ratio of ATP ⁇ S:ATP was 5:5, the RNA bands were more concentrated, indicating that the uniformity of the RNA was better (Fig. 3, c).
  • ATP and modified ATP set the total final concentration of ATP and modified ATP to 1 mM, and use ATP, 50% ATP+50% ATP ⁇ S, 50% ATP+25% ATP ⁇ S+25% 2'OMe-ATP, 50% ATP+25 %ATP ⁇ S+25%2'F-dATP for tailing reaction; in addition, Fluc-A60 mRNA uses ATP, 50% ATP+50% ATP ⁇ S, 50% ATP+50% ATP ⁇ BH 3 , 50% ATP+50% ATP ⁇ CH 3 , 50% ATP+25% ATP ⁇ S+25% ATP ⁇ BH 3 , 50% ATP+25% ATP ⁇ S+25% ATP ⁇ CH 3 for tailing reactions ( FIG. 5 , b).
  • the present invention introduces modified adenosine after the existing poly(A) tail 3' end at the 3' end of the starting RNA molecule to form an extended poly(A) sequence (Fig. 1), which can significantly improve
  • the stability and/or translation efficiency of RNA molecules in cells can achieve the expression of more proteins in cells with less amount of mRNA, effectively reducing the dosage of mRNA used and production costs.
  • Fragment names are: XhoI-A100-RZ-BglII fragment, XhoI-A100-NotIA-RZ-BglII fragment.
  • sequence from the 5' end to the 3' end of the XhoI-A100-RZ-BglII fragment is (SEQ ID NO: 1):
  • the sequence shown by the underlined horizontal line is the XhoI and BglII sites, and the sequence shown by the wavy line underlined is the HDV ribozyme (abbreviated as RZ) sequence.
  • sequence indicated by the underlined horizontal line is XhoI, NotI, BglII site, wherein the sequence indicated by the underlined wavy line
  • the sequence is an HDV ribozyme (abbreviated as RZ) sequence, and there is an A between the NotI and HDV ribozyme sequences.
  • restriction endonucleases XhoI and BglII to excise the above two DNA fragments from the pUC57-simple plasmid provided by GenScript, perform DNA cutting gel recovery, and use it as an insert fragment (Insert); use the constructed T7 Promoter sequence, 5' untranslated region, Kozak sequence, firefly luciferase reporter gene (Fluc) open reading frame, 3' untranslated region of the plasmid, use restriction endonucleases XhoI and BglII to digest, make DNA Gel cutting and recovery to obtain the Fluc vector fragment.
  • T4 DNA ligase to carry out the ligation reaction, connect the XhoI-A100-RZ-BglII fragment and the XhoI-A100-NotIA-RZ-BglII fragment to the Fluc vector fragment respectively, transform with competent cells DH5 ⁇ and Stbl3, and select the single The clones were subjected to sanger sequencing to confirm that the sequences were correct, and plasmids Fluc-A100RZ and Fluc-A100NotIARZ were obtained, respectively.
  • DNA templates required for in vitro transcription in the above plasmids were excised with restriction endonucleases, and recovered by DNA excision gel to obtain 6 kinds of DNA templates.
  • the plasmid Fluc-A100RZ was cut with restriction endonucleases EcoRI and BglII, and the templates Fluc-A100-RZ-DH5 ⁇ and Fluc-A100-RZ-Stbl3 were obtained by DNA cutting gel recovery.
  • T7 RNA polymerase was used for in vitro transcription reaction to obtain corresponding mRNA molecules.
  • in vitro transcription reaction use cap analogs at a final concentration of 1.6 mM and ATP, UTP, CTP at a final concentration of 2 mM, and GTP at a final concentration of 0.4 mM, 20 ng/ul DNA template, 1x T7 reaction buffer, T7 RNA polymerase and RNase inhibitors were reacted at 37°C for 4 hours to terminate the reaction, and DNase I was added to react at 37°C for 15 minutes to remove the DNA template, and then the MEGAclear transcription purification kit (Thermo Fisher) was used to clean the product material is purified.
  • MEGAclear transcription purification kit Thermo Fisher
  • the ribozyme at the 3' end of the synthesized RNA will form adenosine-2', 3'-cyclic phosphate at the 3' end of the RNA after self-cleavage, and T4 can be used at this time
  • T4 can be used at this time
  • the next step of ligation reaction or polymerization reaction is carried out.
  • Fluc for short mRNAs Different firefly luciferase (Fluc for short) mRNAs were used to transfect cells, and an equal amount of renilla luciferase (Rluc for short) reporter gene mRNA was transfected at the same time as a reference.
  • Rluc for short reporter gene mRNA was transfected at the same time as a reference.
  • HEK293T cells when the density of HEK293T cells is about 40%, dilute 0.22 ⁇ l lipofectamine2000 in 50 ⁇ l Opti-MEM, and after standing at room temperature for 5 minutes, add 100 ng Fluc mRNA and 10 ng diluted in 50 ⁇ l Opti-MEM Rluc mRNA, mix well and let stand at room temperature for 20 minutes, finally carefully add to the cells, after 24 hours of incubation, rinse the cells with 1 ⁇ PBS, and then use an appropriate amount of lysis buffer (100mM potassium phosphate [pH 7.8], 0.2% ( v/v) Triton X-100) lyse the cells, centrifuge at 10,000rpm at room temperature for 1 minute, take an appropriate amount of supernatant, add the corresponding volume of the luciferase reaction substrate provided in the dual luciferase reporter gene detection kit, and use the multifunctional Fluorescence values were recorded on a microplate reader.
  • lysis buffer 100mM potassium phosphate [
  • Dividing the fluorescence value of firefly luciferase by the fluorescence value of Renilla luciferase is the expression of the reporter gene, which is used as a reference value for the protein expression efficiency of mRNA.
  • the present invention introduces a modified hepatitis D virus ribozyme (HDV RZ) sequence (Fig. 7, a) at the 3' end of the poly(A) sequence of the plasmid vector, after the in vitro transcription reaction generates the mRNA molecule, the 3' end
  • the ribozyme performs self-cleavage to form a poly(A) tail with a fixed length and prevents the residue of non-A bases at the 3' end of poly(A) (Fig. 7, b).
  • the constructed Fluc-A100-RZ plasmid and Fluc-A100-NotIA-RZ plasmid were respectively transferred into competent cells of strains DH5a and Stbl3 for plasmid amplification, and the mRNAs synthesized by in vitro transcription of the extracted plasmids were labeled as: RZ-D, RZ-S; NotIA-RZ-D, NotIA-RZ-S; NotIcut-D, NotIcut-S, where the suffix -D represents the clone from the DH5a strain, and the suffix -S represents the clone from the Stbl3 strain.
  • RNA bands appeared at about 100bp, that is, ribozyme self-cutting bands ( Figure 9, a), and the mRNA can be purified after purification Complete removal of the ribozyme molecule will not have any impact on subsequent reactions and cell experiments.

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Abstract

一种提高RNA分子的胞内翻译效率和/或稳定性的方法。提供RNA分子的制备方法，包括在起始RNA分子的poly(A)尾的3'端添加由修饰腺苷酸和任选的未修饰腺苷酸组成的修饰poly(A)序列的步骤。该RNA分子可以是mRNA分子。还提供RNA分子、药物组合物、相应的用途以及试剂盒。该方法制备得到的RNA分子的poly(A)尾得以延长并在延长的部分具有修饰的腺苷酸，相对于poly(A)尾未延长的起始RNA分子或poly(A)尾延长部分不包含修饰腺苷酸的RNA分子，该RNA分子具有提升的胞内翻译效率和/或稳定性。

Description

提高RNA分子的胞内翻译效率和/或稳定性的方法 技术领域
本发明涉及提高RNA分子的胞内翻译效率和稳定性的方法。
背景技术
体外合成的mRNA通过电穿孔、脂质体等递送技术被递送到细胞内，可用于在细胞水平和动物体内翻译表达任何天然或非天然的蛋白质产物，包括微生物多肽抗原、肿瘤抗原多肽的串联物、抗体、嵌合抗原受体、天然及突变形式的酶和细胞因子等。目前，利用体外合成的mRNA在细胞内表达蛋白质的方法已被广泛应用于多种传染病疫苗的开发，如已获批的新冠疫苗、进入三期临床试验的流感病毒疫苗、呼吸道合胞病毒疫苗和巨细胞病毒疫苗等。同时，mRNA技术也在肿瘤免疫治疗和罕见病治疗领域崭露头角，目前有多个mRNA药物进入一期或二期临床试验。
大量研究证明优化mRNA分子结构、引入核苷酸修饰和改进纯化方法等，可极大地提升mRNA的稳定性和翻译效率、降低mRNA免疫原性〔Pardi，N.，Hogan，M.J.，Porter，F.W.，&Weissman，D.(2018).mRNA vaccines-a new era in vaccinology.Nature reviews.Drug discovery，17(4)，261-279〕。Katalin Karikó与Drew Weissman因在mRNA分子中创新地引入假尿嘧啶核苷(pseudouridine，ψ)修饰而摘得2021年的“诺奖风向标”拉斯克奖。但现在已应用于mRNA技术的核苷酸修饰，例如假尿嘧啶核苷(ψ)和5-甲基胞嘧啶核苷(5-methylcytosine，m5C)修饰，都是针对mRNA poly(A)以外的区域或整条mRNA〔Pardi，N.，Hogan，M.J.，Porter，F.W.，&Weissman，D.(2018).mRNA vaccines-a new era in vaccinology.Nature reviews.Drug discovery，17(4)，261-279〕。poly(A)作为mRNA分子3’端的重要结构，在mRNA的翻译效率和稳定性方面发挥了关键作用〔Passmore，L.A.，&Coller，J.(2021).Roles of mRNA poly(A)tails in regulation  of eukaryotic gene expression.Nature reviews.Molecular cell biology，10.1038/s41580-021-00417-y.Advance onlinepublication〕。但目前已报道的研究仅止步于优化poly(A)的长度，poly(A)中引入核苷酸修饰对于mRNA的稳定性和翻译效率的影响尚未得到充分探索。
在核苷酸修饰中，除了假尿嘧啶核苷(ψ)修饰这样的碱基修饰，还有针对磷酸基、糖基的修饰，例如以硫代磷酸(phosphorothioate，PS)修饰、硼代磷酸(boranophosphate，PB)修饰、甲基代磷酸(methylphosphonate，MP)修饰为代表的磷酸基修饰和以锁核酸(locked nucleic acid，LNA)修饰、2’甲氧基(2’-O-Methyl，2’OMe)修饰、2’氟代(2’-Fluoro，2’F)修饰为代表的糖基修饰。这些修饰能极大地提高RNA的稳定性，并已经成功应用于已上市的多款反义寡核苷酸(antisense oligonucleotides，ASO)药物和小干扰RNA(small interfering RNA，siRNA)药物〔Duffy，K.，Arangundy-Franklin，S.，&Holliger，P.(2020).Modified nucleic acids：replication，evolution，and next-generation therapeutics.BMC biology，18(1)，112〕。这些核苷酸修饰是否能用于合成mRNA并提高mRNA的蛋白质表达效率，尚未得到充分研究。
发明内容
本发明第一方面提供一种RNA分子的制备方法，所述方法包括在起始RNA分子的poly(A)尾的3’端添加由修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸组成的修饰poly(A)序列的步骤。
在一个或多个实施方案中，所述方法用于制备翻译效率和/或稳定性提高的RNA分子。
在一个或多个实施方案中，所述poly(A)尾为长度为30-250个、优选50-200个、更优选60-200个、更优选60-150个连续的含有未修饰腺苷酸并任选含有修饰腺苷酸的腺苷酸序列1，所述修饰poly(A)序列为长度为1-250个、优选10-250个、更优选20-200个、更优选30-200个核苷酸的腺苷酸序列2；优选地，所述腺苷酸序列2中至少1％、优选至少5％、优选10-100％、更优选20-100％、更优选50-100％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸。
在一个或多个实施方案中，采用连接反应或聚合反应在所述起始RNA分子的所述poly(A)尾3’端添加所述修饰poly(A)序列。
在一个或多个实施方案中，所述连接反应中，使用RNA连接酶将所述修饰poly(A)序列与所述poly(A)尾连接；优选地，所述修饰poly(A)序列的5’端具有5’P修饰，3’端具有选自以下的修饰：3’ddC、3’BHQ-1、3’BHQ-2、3’MGB、3’Dabcul、3’6-TAMRA、3’6-FAM、3’CY5、3’CY3、3’6-ROX、3’inverted dT、3’Biotin、3’Biotin-TEG、3’Amino modified C7、3’Phosphate、3’Digoxigenin、3’Thiol modifier C6 S-S、3’Thiol modifier C3 S-S、3’Spacer C3、3’Spacer C6、3’Spacer 9、3’Spacer 18、3’dSpacer、3’Cholesteryl-TEG和3’Ferrocene dT。
在一个或多个实施方案中，所述聚合反应中，使用聚合酶或末端转移酶以修饰ATP及任选的未修饰ATP为底物，在起始RNA分子的所述poly(A)尾的3’端添加修饰腺苷酸和任选的未修饰腺苷酸；优选地，聚合反应的反应体系不含有未修饰ATP，或含有未修饰ATP时，修饰ATP和未修饰ATP的比例在1∶19到19∶1的范围内，如1∶9到9∶1或2∶8到8∶2。
在一个或多个实施方案中，所述起始RNA分子含有修饰的核苷酸。
在一个或多个实施方案中，所述方法还包括通过DNA模板制备具有所述poly(A)尾的起始RNA分子的步骤；优选地，所述DNA模板从5’端到3’端含有依次连接的启动子序列、5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和poly(A)尾序列；更优选地，所述DNA模板在该poly(A)尾之后还直接连接有核酶序列；优选地，所述核酶是具有自剪切功能的核酶，优选选自HDV核酶、发夹状核酶和锤头状核酶。
在一个或多个实施方案中，所述修饰腺苷酸和修饰胞苷酸中的修饰包括针对磷酸基的修饰和针对糖基的修饰；优选地，所述修饰选自硫代磷酸(PS)修饰、硼代磷酸(PB)修饰、甲基代磷酸(MP)修饰、锁核酸(LNA)修饰、2’甲氧基(2’OMe)修饰和2’氟代(2’F)修饰；优选地，所述修饰腺苷酸为PS修饰的腺苷酸、PB修饰的腺苷酸、MP修饰的腺苷酸、LNA修饰的腺苷酸、PS修饰的腺苷酸与2’OMe修饰的腺苷酸的混合物、PS修饰的腺苷酸与PB修饰的腺苷酸、PS修饰的腺苷酸与MP修饰的腺苷酸、或PS修饰的腺苷酸与2’F 修饰的腺苷酸的混合物，所述修饰胞苷酸为LNA修饰的胞苷酸。
本发明第二方面提供一种mRNA分子的制备方法，所述方法包括以下步骤：
(1)使用DNA模板通过体外转录制备得到具有poly(A)尾的mRNA分子；和
(2)采用连接反应或聚合反应在步骤(1)获得的mRNA分子的所述poly(A)尾的3’端添加修饰腺苷酸和/或修饰胞苷酸和任选的未修饰腺苷酸或含有所述修饰腺苷酸和/或修饰胞苷酸和任选的未修饰腺苷酸的修饰的poly(A)序列，从而制备得到所述mRNA分子。
在一个或多个实施方案中，步骤(2)中，以修饰ATP及任选的未修饰ATP为底物，使用聚合酶或末端转移酶在步骤(1)获得的mRNA分子的所述poly(A)尾的3’端添加修饰腺苷酸和任选的未修饰腺苷酸，从而制备得到所述mRNA分子。
在一个或多个实施方案中，步骤(1)中，所述DNA模板从5’端到3’端含有依次连接的启动子序列、5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和poly(A)尾；更优选地，所述DNA模板在该poly(A)尾之后还直接连接有核酶序列；优选地，所述核酶是具有自剪切功能的核酶，优选选自HDV核酶、发夹状核酶和锤头状核酶。
在一个或多个实施方案中，步骤(2)的反应体系中不含有未修饰ATP，或含有修饰ATP和未修饰ATP时，其中修饰ATP和未修饰ATP的比例在1∶19到19∶1的范围内，如1∶9到9∶1或2∶8到8∶2。
在一个或多个实施方案中，所述连接反应中，使用RNA连接酶将含有所述修饰腺苷酸和/或修饰胞苷酸和任选的未修饰腺苷酸的修饰poly(A)序列与所述mRNA分子的所述poly(A)尾的3’端连接；优选地，所述修饰poly(A)序列的5’端具有5’P修饰，3’端具有选自以下的修饰：3’ddC、3’BHQ-1、3’BHQ-2、3’MGB、3’Dabcul、3’6-TAMRA、3’6-FAM、3’CY5、3’CY3、3’6-ROX、3’inverted dT、3’Biotin、3’Biotin-TEG、3’Amino modified C7、3’Phosphate、3’Digoxigenin、3’Thiol modifier C6 S-S、3’Thiol modifier C3 S-S、3’Spacer C3、3’Spacer C6、 3’Spacer 9、3’Spacer 18、3’dSpacer、3’Cholesteryl-TEG和3’Ferrocene dT。
在一个或多个实施方案中，所述聚合反应中，反应体系不含有未修饰ATP，或含有未修饰ATP时，修饰ATP和未修饰ATP的比例在1∶19到19∶1的范围内，如1∶9到9∶1或2∶8到8∶2。
本发明第三方面提供一种RNA分子，该RNA分子具有含有多个未修饰的腺苷酸和一个或多个修饰的腺苷酸和/或一个或多个修饰的胞苷酸的poly(A)尾；其中，该poly(A)尾具有长度为30-250个、优选50-200个、更优选60-200个、更优选60-150个连续核苷酸的腺苷酸序列1和与该腺苷酸序列1的3’端连接的长度为1-250个、优选10-250个、更优选20-200个、更优选30-200个连续核苷酸的腺苷酸序列2；其中，该腺苷酸序列1由未修饰的腺苷酸组成，或者含有未修饰的腺苷酸和修饰的腺苷酸，该腺苷酸序列2含有至少一个修饰的腺苷酸和/或至少一个修饰的胞苷酸；其中，该腺苷酸序列1含有修饰的腺苷酸时，硫代腺苷不是唯一的修饰腺苷酸，优选地，该腺苷酸序列1中不含有硫代腺苷修饰。
在一个或多个实施方案中，所述RNA分子选自mRNA分子、长链非编码RNA或非编码小RNA前体。
在一个或多个实施方案中，该腺苷酸序列2中至少1％、优选至少5％、优选至少10％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸；优选地，该腺苷酸序列2中优选10-100％、更优选20-100％、更优选50-100％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸。
在一个或多个实施方案中，所述腺苷酸序列1由未修饰的腺苷酸组成，所述腺苷酸序列2含有修饰的腺苷酸和/或修饰的胞苷酸与任选的未修饰的腺苷酸，且该腺苷酸序列2与腺苷酸序列1的3’端连接的第1个核苷酸是修饰的腺苷酸。
在一个或多个实施方案中，所述修饰腺苷酸和修饰胞苷酸中的修饰包括针对磷酸基的修饰和针对糖基的修饰；优选地，所述修饰选自硫代磷酸(PS)修饰、硼代磷酸(PB)修饰、甲基代磷酸(MP)修饰、锁核酸(LNA)修饰、2’甲氧基(2’OMe)修饰和2’氟代(2’F)修饰；优选地，所述修饰腺苷酸为PS 修饰的腺苷酸、PB修饰的腺苷酸、MP修饰的腺苷酸、LNA修饰的腺苷酸、PS修饰的腺苷酸与2’OMe修饰的腺苷酸的混合物、PS修饰的腺苷酸与PB修饰的腺苷酸、PS修饰的腺苷酸与MP修饰的腺苷酸、或PS修饰的腺苷酸与2’F修饰的腺苷酸的混合物，所述修饰胞苷酸为LNA修饰的胞苷酸。
在一个或多个实施方案中，所述mRNA分子从5’到3’含有依次连接的5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和所述poly(A)分子。
在一个或多个实施方案中，所述开放阅读框编码感兴趣的蛋白质或多肽分子；优选地，所述蛋白质或多肽分子选自病原体抗原、肿瘤抗原、细胞因子、激素、抗体、嵌合抗原受体、酶和结构蛋白。
在一个或多个实施方案中，所述病原体选自病毒、细菌、真菌、螺旋体和寄生虫。
在一个或多个实施方案中，所述肿瘤抗原为肿瘤相关性抗原和肿瘤特异性抗原。
本发明第四方面提供一种药物组合物，所述药物组合物含有本文任一实施方案所述的RNA分子或采用本文任一实施方案所述的方法制备得到的RNA分子或mRNA分子和药学上可接受的载体。
在一个或多个实施方案中，所述药物组合物是疫苗，优选为预防性疫苗或治疗性疫苗，其中，预防性疫苗中所述RNA分子编码抗原多肽，治疗性疫苗中所述RNA分子编码治疗性多肽。
本发明第五方面提供选自以下的应用：
(1)修饰的ATP、修饰的CTP、修饰ATP和/或修饰的CTP与未修饰的ATP的混合物、或含修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸的poly(A)分子在制备胞翻译效率和/或稳定性提高的已有poly(A)尾的RNA分子中的应用，或修饰的腺苷酸和/或修饰的胞苷酸或由修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子在提高已有poly(A)尾的RNA分子的胞内翻译效率和/或稳定性中的应用；优选地，所述修饰选自硫代磷酸修饰、硼代磷酸修饰、甲基代磷酸修饰、锁核酸修饰、2’甲氧基修饰和2’氟代修饰；
(2)本文任一实施方案所述的RNA分子或采用本文任一实施方案所述的方法制备得到的RNA分子或mRNA分子在制备核酸药物或核酸疫苗中的应用。
在一个或多个实施方案中，所述修饰ATP和/或修饰的CTP与未修饰ATP的混合物中，修饰的ATP和/或修饰的CTP和未修饰ATP的比例在1∶19到19∶1的范围内，如在1∶9到9∶1或2∶8到8∶2的范围内。
在一个或多个实施方案中，所述由修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子不含有未修饰腺苷酸，或含有时，修饰的修饰腺苷酸和/或修饰的胞苷酸与未修饰的腺苷酸的比例在1∶19到19∶1的范围内，如在1∶9到9∶1或2∶8到8∶2的范围内。
本发明第六方面提供一种试剂盒，所述试剂盒含有修饰ATP和/或修饰的CTP与未修饰ATP的混合物，或含有由修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子，以及实施连接反应或聚合反应所需的试剂。
在一个或多个实施方案中，所述修饰ATP和/或修饰的CTP与未修饰ATP的混合物中，修饰的ATP和/或修饰的CTP与未修饰ATP的比例在1∶19到19∶1的范围内，如在1∶9到9∶1或2∶8到8∶2的范围内；所述由修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子不含有未修饰腺苷酸，或含有时，修饰腺苷酸和/或修饰胞苷酸与未修饰腺苷酸的比例在1∶19到19∶1的范围内，如在1∶9到9∶1或2∶8到8∶2的范围内。
在一个或多个实施方案中，所述试剂盒用于实施本文任一实施方案所述的RNA制备方法或mRNA制备方法。
附图说明
图1：本发明poly(A)分子的示意图。poly(A)长度为31-500个腺苷酸，含有多个未修饰的腺苷酸和一个或多个修饰的腺苷酸；其中，该poly(A)分子的5’端为30-250个连续的未修饰的腺苷酸的腺苷酸序列1，该腺苷酸序列1紧连着修饰比例≥1％、长度为1-250nt的腺苷酸序列2。符号“＊”、“+”代表通过 连接反应或聚合反应引入的核苷酸修饰。
图2：通过连接反应在Fluc-A60 mRNA 3’端添加含硫代磷酸(phosphorothioate，PS)修饰腺苷酸和锁核酸(locked nucleic acid，LNA)修饰腺苷酸的寡聚核苷酸(RNA oligonucleotides，简写为RNA oligo)可提高mRNA在细胞内的蛋白质表达效率。(a)整合进RNA分子的无修饰腺苷酸、PS修饰腺苷酸、LNA修饰腺苷酸和LNA修饰胞苷酸的结构图。符号“＊”代表PS修饰，符号“+”代表LNA修饰。(b)实验流程示意图：将含3个PS修饰位点或2个LNA修饰位点的寡聚核苷酸连接到Fluc-A60 mRNA 3’端。符号“＊”代表PS修饰，符号“+”代表LNA修饰。(c)连接反应得到的Fluc A20 mRNA、Fluc A20-PS mRNA转染HEK293T细胞后不同时间点的蛋白质表达效率比较。(d)连接反应得到的Fluc A20 mRNA、Fluc A20-LNA mRNA转染HEK293T细胞后不同时间点的蛋白质表达效率比较。(e)连接反应得到的Fluc A20 mRNA、Fluc A20-LNAC mRNA转染HEK293T细胞后不同时间点的蛋白质表达效率比较。
图3：使用E.coli poly(A)聚合酶和硫代磷酸(PS)修饰的ATP(Adenosine-5′-O-(1-Thiotriphosphate)，1-Thio-ATP，ATPαS)在70bp RNA片段的3’端添加PS修饰腺苷酸。(a)PS修饰的ATP(ATPαS)与ATP的结构图。符号“＊”代表PS修饰。(b)使用ATPαS在70nt RNA片段3’端添加PS修饰腺苷酸反应的实验流程示意图。符号“＊”代表PS修饰。(c)ATPαS∶ATP混合比例分别为0∶10、1∶9、2∶8、3∶7、4∶6、5∶5、6∶4、7∶3、8∶2、9∶1、10∶0、0∶0，使用E.coli poly(A)聚合酶反应为70bp RNA片段加poly(A)尾，反应产物用琼脂糖凝胶电泳进行检测。
图4：使用E.coli poly(A)聚合酶和硫代磷酸(PS)修饰的ATP(ATPαS)在Fluc-A60和Fluc-A0 mRNA的3’端添加PS修饰腺苷酸可提高mRNA在细胞内的表达效率。(a)使用ATPαS在mRNA片段3’端添加PS修饰腺苷酸反应的实验流程示意图。符号“＊”代表PS修饰。(b)将ATP与ATPαS的总终浓度分别固定为0.1mM、0.3mM、1mM，将ATPαS∶ATP分别按照0∶10、2∶8、5∶5、8∶2、10∶0、0∶0的比例混合，为Fluc-A60和Fluc-A0mRNA加poly(A)尾， 反应产物用琼脂糖凝胶电泳进行检测。(c)不同ATP与ATPαS的总终浓度、不同ATPαS∶ATP比例体外转录制备获得的mRNA在细胞内的蛋白质表达效率比较。(d)不含poly(A)、含长度为A60的poly(A)的mRNA，分别使用ATP、(ATP+ATPαS)进行加尾反应，比较同样质量的mRNA产物在细胞内的蛋白质表达效率。
图5：使用E.coli poly(A)聚合酶、硫代磷酸(PS)修饰的ATP(ATPαS)、2’甲氧基修饰的ATP(2’OMe-ATP)、2’氟代修饰的ATP(2’F-dATP)、硼代磷酸(PB)修饰的ATP(ATPαBH3)与甲基代磷酸(MP)修饰的ATP(ATPαCH3)在Fluc-A60 mRNA的3’端添加修饰腺苷酸可提高mRNA在细胞内的表达效率。(a)2’甲氧基修饰ATP、2’氟代修饰ATP、硼代磷酸修饰ATP与甲基代磷酸修饰ATP的结构图。符号“#”代表2’甲氧基修饰或2’氟代修饰。符号“＊”代表PS修饰、PB修饰或MP修饰。(b)在mRNA片段3’端添加多种修饰腺苷酸反应的实验流程示意图。符号“＊”代表PS修饰、PB修饰或MP修饰，符号“#”代表2’甲氧基修饰或2’氟代修饰。(c)mRNA分别使用ATP、50％ATP+50％ATPαS、50％ATP+25％ATPαS+25％2’OMe-ATP、50％ATP+25％ATPαS+25％2’F-dATP进行加尾反应，比较同样质量的mRNA产物在细胞内的蛋白质表达效率。(d)mRNA分别使用ATP、50％ATP+50％ATPαS、50％ATP+50％ATPαBH3、50％ATP+50％ATPαCH3、50％ATP+25％ATPαS+25％ATPαBH3、50％ATP+25％ATPαS+25％ATPαCH3进行加尾反应，比较同样质量的mRNA产物在细胞内的蛋白质表达效率。
图6：使用E.coli poly(A)聚合酶和硫代磷酸(PS)修饰的ATP(ATPαS)在假尿嘧啶核苷修饰、2’甲氧基腺苷修饰或2’氟代腺苷修饰的Fluc-A60 mRNA的3’端添加PS修饰腺苷酸可提高mRNA在细胞内的蛋白质表达效率。(a)使用ATPαS在假尿嘧啶核苷修饰的mRNA片段3’端添加PS修饰腺苷酸反应的实验流程示意图。符号“＊”代表PS修饰，符号“ψ”代表假尿嘧啶核苷修饰。(b)在假尿嘧啶核苷修饰的mRNA片段的3’端添加PS修饰腺苷酸，比较同样质量的mRNA产物在细胞内的蛋白质表达效率。(c)使用ATPαS在2’甲氧基腺苷或2’氟代腺苷修饰的mRNA片段3’端添加PS修饰腺苷酸反应的 实验流程示意图。符号“＊”代表PS修饰，符号“#”代表2’甲氧基修饰或2’氟代修饰。(d)在2’甲氧基腺苷修饰的mRNA片段的3’端添加PS修饰腺苷酸，比较同样质量的mRNA产物在细胞内的蛋白质表达效率。(e)在2’氟代腺苷修饰的mRNA片段的3’端添加PS修饰腺苷酸，比较同样质量的mRNA产物在细胞内的蛋白质表达效率。
图7：本发明体外转录模板及HDV核酶结构。(a)HDV核酶的二级结构与序列。(b)使用新型体外转录模板制备mRNA的过程示意图。在质粒载体的poly(A)序列3’端是HDV核酶序列，使用此质粒载体制备线性化的DNA模板进行体外转录反应，得到的mRNA的3’端核酶进行自剪切，形成长度均一、无非A碱基残留的均一的poly(A)尾。
图8：采用三种方案构建质粒载体、制备体外转录模板、合成mRNA。(a)使用Fluc-A100-RZ质粒制备DNA模板，经体外转录合成RZ mRNA。(b)使用Fluc-A100-NotIA-RZ质粒制备DNA模板，经体外转录合成NotIA-RZ mRNA。(c)使用Fluc-A100-NotIA-RZ质粒，使用酶切位点NotI制备DNA模板，经体外转录合成NotIcut mRNA。
图9：检测不同mRNA的电泳条带及蛋白质表达效率。(a)纯化前的RZ、NotIA-RZ、NotIcut用6％PAGE变性胶进行电泳，可观察到核酶自剪切条带(黑色三角形标注)。(b-d)RZ、NotIA-RZ、NotIcut分别在HEK293T、HCT116、Hela细胞中的蛋白质表达效率。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
poly(A)是mRNA分子3’端的重要结构。研究表明，在细胞内，mRNA的poly(A)尾与poly(A)结合蛋白PABP结合，PABP与EIF4G复合物相互作用，对于mRNA的稳定性和翻译均至关重要。本发明发现，在起始RNA分子3’端已有的poly(A)尾3’端后引入修饰的腺苷酸、修饰的胞苷酸或修饰的腺苷酸与修饰的胞苷酸，形成延长的poly(A)序列，由此获得的RNA分子在细胞内具有 显著提升的翻译效率和/或稳定性。
因此，本发明提供一种制备RNA分子的方法。采用该方法制备得到的RNA分子在细胞内具有提升的翻译效率和/或稳定性。该方法包括在起始RNA分子已有的poly(A)尾的3’端添加含有修饰腺苷酸和/或修饰胞苷酸的修饰poly(A)序列的步骤。由此制备得到的RNA分子相对于该起始RNA分子具有延长的poly(A)尾，且其延长的部分含有修饰腺苷酸和/或修饰胞苷酸，活性上比该起始RNA分子或poly(A)尾延长部分不包含修饰腺苷酸和/或修饰胞苷酸的RNA分子具有提升的胞内翻译效率和/或稳定性。
本文中，RNA的稳定性(RNA stability)指RNA分子抗拒降解的程度，稳定性高的RNA不易发生降解，具有较长的半衰期。
本文中，修饰的腺苷酸(adenosine monophosphate，AMP)或修饰的胞苷酸(CMP)指通过化学合成或使用聚合反应的方法整合进RNA分子中的修饰的单磷酸腺苷或单磷酸胞苷。
本文中，修饰的腺苷酸和修饰的胞苷酸中的修饰包括针对磷酸基的修饰和针对糖基的修饰。针对磷酸基的修饰包括但不限于硫代磷酸(phosphorothioate，PS)修饰、硼代磷酸(boranophosphate，PB)修饰和甲基代磷酸(methylphosphonate，MP)修饰。针对糖基的修饰包括但不限于锁核酸(locked nucleic acid，LNA)修饰、2’甲氧基(2’-O-Methyl，2’OMe)修饰和2’氟代(2’-Fluoro，2’F)修饰。示例性的PS修饰和LNA修饰如图2(b)和3(a)所示，示例性的2’OMe修饰和2’F修饰如图5(a)所示。在本文各实施方案的优选实施例中，延长的poly(A)尾中的修饰腺苷酸包括PS修饰的腺苷酸、LNA修饰的腺苷酸和LNA修饰的胞苷酸中的至少一种。
本文中，所述起始RNA分子指本身已在3’端具有poly(A)尾的且待延长其poly(A)尾的RNA分子，与之相对应的是制备得到的具有延长的poly(A)尾的胞内翻译效率和/或稳定性提升的“RNA分子”(也可称为“具有延长的poly(A)尾的RNA分子”)。
本文中，起始RNA分子可以是本领域周知的任意RNA分子，包括但不限于mRNA分子、长链非编码RNA(lncRNA)或非编码小RNA前体(如miRNA 或siRNA前体)。起始RNA分子可以是具有任何感兴趣用途的RNA分子，包括但不限于疾病的诊断、预防和治疗用途。
在一些实施方案中，该起始RNA分子可以为mRNA分子，从5’到3’含有依次连接的5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和poly(A)尾。通常，起始mRNA分子的poly(A)尾的长度可为30-250个腺苷酸，如50-200个腺苷酸、60-200个腺苷酸或60-150个腺苷酸。
本文中，5’非翻译区(5’UTR)指位于开放阅读框上游不被翻译为蛋白质的区域。Kozak序列指真核生物mRNA 5’端的一段序列，在翻译起始中发挥重要作用。3’非翻译区(3’UTR)指位于开放阅读框下游不被翻译为蛋白质的区域。5’UTR和3’UTR用于调节mRNA翻译、半衰期和亚细胞定位。可选择使用本领域周知的常用于mRNA制备的5’UTR、Kozak序列和3’UTR来实施本发明。通常，来自高表达基因(如α和β珠蛋白基因)的天然UTR是合成mRNA的首选。此外，UTR可根据细胞类型进行优化，如通过去除3’UTR中的miRNA结合位点和富含AU的区域，将mRNA降解降至最低。
本文中，所述开放阅读框编码感兴趣的蛋白质或多肽分子。本文中，蛋白质或多肽分子包括但不限于病原体抗原、肿瘤抗原、细胞因子、激素、抗体、嵌合抗原受体、酶和结构蛋白。
本文中，所述病原体包括但不限于病毒、细菌、真菌、螺旋体和寄生虫等。病原体抗原可以是来自这些病原体的具有免疫原性的多肽，包括本领域周知的用做疫苗以引发免疫原性反应的抗原肽。
在一些实施方案中，所述蛋白质或多肽分子是抗原多肽。在一些实施方案中，所述抗原多肽来源于病原体。本文中，病原体指能够造成人或动植物感染疾病的微生物。在一些实施方案中，所述病原体包括但不限于病毒、细菌、真菌、原生动物和/或寄生虫。
在一些实施方案中，所述病原体包括但不限于以下的病毒：新冠病毒、腺病毒、单纯疱疹、脑炎病毒、乳头瘤病毒、水痘-带状疱疹病毒人巨细胞病毒、人疱疹病毒、人乳头瘤病毒、脊髓灰质炎病毒、乙型肝炎病毒、诺沃克病毒、柯萨奇病毒、甲型肝炎病毒、脊髓灰质炎病毒、严重急性呼吸道综合征病毒、 丙型肝炎病毒、黄热病毒、登革热病毒、西尼罗病毒、风疹病毒、戊型肝炎病毒、人免疫缺陷病毒、流感病毒、瓜纳里托病毒、胡宁病毒、拉沙病毒、马丘波病毒、萨比亚病毒、埃博拉病毒、马尔堡病毒、麻疹病毒、腮腺炎病毒、副流感病毒、呼吸道合胞病毒、亨德拉病毒、尼帕病毒、狂犬病病毒、丁型肝炎病毒、轮状病毒、环状病毒、科罗拉多壁虱热病毒、版纳病毒、人肠道病毒、汉坦病毒、西尼罗病毒、中东呼吸道综合征冠状病毒、日本脑炎病毒和水疱性疱疹病毒。
在一些实施方案中，所述蛋白质或多肽分子是肿瘤抗原。例如肿瘤相关性抗原(tumor-associated antigens)和肿瘤特异性抗原(tumor-specific antigens，也称为肿瘤新生抗原neoantigen)。
在一些实施方案中，所述肿瘤相关抗原是正常细胞和肿瘤细胞上都存在的抗原分子，并非肿瘤细胞所特有，它通常在肿瘤细胞增殖时出现高表达状态。示例性的肿瘤相关抗原包括：胚胎抗原、糖蛋白抗原、鳞状细胞抗原等。更具体而言，肿瘤相关抗原包括但不限于甲胎蛋白(AFP)、癌胚抗原(CEA)、纽约食管鳞状上皮癌抗原1(NY-ESO-1)、黑色素瘤相关抗原A3(MAGE-A3)和酪氨酸酶等。在一些实施方案中，肿瘤新生抗原由肿瘤细胞的非同义突变所产生，仅表达于肿瘤细胞表面，不存在于任何不同发育阶段的正常细胞上。
本文所述的细胞因子、激素、抗体、酶和结构蛋白可以是本领域周知的具有治疗活性或预防活性的蛋白或多肽，包括天然蛋白或功能性肽段，也包括优化突变的蛋白。
本文所述的嵌合抗原受体(CAR)可以是目前已知的针对各种抗原(尤其是肿瘤相关抗原)的CAR。示例性的抗原包括但不限于CD19、CD20、MUC1、CD22、CD23、CD30、CD33、CD44v7/8、CD70、VEGFR1、VEGFR2、MSLN、CA125、PD1、PD-L1、FAP和EGFR等。CAR的结构为本领域所周知，示例性的CAR通常包括信号肽、抗体序列、铰链区、跨膜区以及胞内信号转导区等。
在一些实施方案中，所述开放阅读框是密码子优化的。
本文中，起始RNA分子中可含有修饰的核苷酸。本领域周知的修饰的核 苷酸均可包含于起始RNA分子中。同样地，修饰包括针对磷酸基的修饰和/或针对糖基的修饰。修饰的核苷酸可以是修饰的腺苷酸、修饰的鸟苷酸、修饰的胞苷酸和修饰的尿苷酸。针对磷酸基的修饰包括但不限于硫代磷酸(phosphorothioate，PS)修饰、硼代磷酸(boranophosphate，PB)修饰、甲基代磷酸(methylphosphonate，MP)修饰、甲磺酰基-氨基磷酸(mesyl-phosphoramidate，MsPA)修饰。针对糖基的修饰包括但不限于锁核酸(locked nucleic acid，LNA)修饰、限制性乙基桥接核酸(constrained ethyl bridged nucleic acid，cEt)修饰、乙烯桥接核酸(ethylene-bridged nucleic acid，ENA)修饰；2’甲氧基(2’-O-methyl，2’OMe)修饰、2’氟代(2’-fluoro，2’F)修饰、2’-氧-甲氧乙基(2’-O-methoxyethyl，2’MOE)修饰。针对碱基的修饰包括但不限于甲基化修饰、氟代修饰、硫代修饰、溴代修饰。起始RNA分子中的示例性修饰包括但不限于假尿苷、5-甲基尿苷、N1-甲基假尿苷、2-硫代尿苷、4-硫代尿苷、5-甲氧基尿苷、5-甲基胞苷、2’-氟代尿苷、2’-氟代鸟苷、5-溴代尿苷、α-硫代胞苷、N1-甲基腺苷、5-甲基胞苷以及硫代磷酸修饰的腺苷酸、锁核酸修饰的腺苷、2’甲氧基腺苷和2’氟代腺苷等。示例性的修饰的核苷、核苷酸可参见CN103974724A和Roberts，T.C.等，〔2020，“Advances in oligonucleotide drug delivery.Nature reviews”，Drug discovery，19(10)，673-694〕，本文以引用的方式将其全文纳入本文。
应理解的是，修饰的核苷酸不需要沿着起始RNA分子的整个长度进行均匀的分布。在起始RNA分子的各个位置可存在不同的核苷酸修饰。修饰的核苷酸可存在于起始RNA分子的任何包括，例如，对于起始mRNA分子，可在其5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和poly(A)尾中的任意一个或多个区域存在任意一种或多种修饰核苷酸。
本文中，可采用通过连接反应或聚合反应在起始RNA分子已有的poly(A)尾的3’端添加含有修饰腺苷酸和/或修饰胞苷酸的修饰poly(A)序列。
可采用本领域熟知的方法实施所述连接反应或聚合反应。示例性的连接反应包括，使用RNA连接酶将本文所述的含有修饰腺苷酸和/或修饰胞苷酸的寡核苷酸序列(RNA oligonucleotides，简写为RNA oligo)与起始RNA分子已有 的poly(A)尾连接。该寡核苷酸序列含有一个或多个修饰腺苷酸和/或修饰胞苷酸。在一些实施方案中，该寡核苷酸序列由修饰腺苷酸和/或修饰胞苷酸组成。在另外一些实施方案中，该寡核苷酸序列由修饰腺苷酸和/或修饰胞苷酸与未修饰腺苷酸组成。在这些实施方案中，寡核苷酸序列中，修饰腺苷酸和/或修饰胞苷酸的含量至少为1％，优选至少为5％，优选至少为10％。在一些实施方案中，寡核苷酸序列中，修饰腺苷酸和/或修饰胞苷酸的比例为5-100％，如10-100％、20-100％、30-100％、40-100％、50-100％等，或者可为5-60％、10-60％、15-60％或20-50％等。在一些实施方案中，所述寡核苷酸序列为本文任一实施方案所述的腺苷酸序列2。
通常，为了保证连接效率，该寡核苷酸序列的5’端可具有5’P修饰，3’端可具有选自以下的修饰：3’ddC、3’BHQ-1、3’BHQ-2、3’MGB、3’Dabcul、3’6-TAMRA、3’6-FAM、3’CY5、3’CY3、3’6-ROX、3’inverted dT、3’Biotin、3’Biotin-TEG、3’Amino modified C7、3’Phosphate、3’Digoxigenin、3’Thiol modifier C6 S-S、3’Thiol modifier C3 S-S、3’Spacer C3、3’Spacer C6、3’Spacer 9、3’Spacer 18、3’dSpacer、3’Cholesteryl-TEG、3’Ferrocene dT。
连接反应的反应体系中可含有起始RNA分子、所述寡核苷酸序列、ATP、反应缓冲液、连接酶、RNA酶抑制剂等。具体的反应条件可以是常规的反应条件。
在RNA 3’端添加腺苷酸和/或胞苷酸的聚合反应中，可以用修饰ATP和/或修饰CTP为底物，使用酶在mRNA分子的poly(A)的3’端添加未修饰核苷酸和修饰核苷酸。可用于本发明在RNA 3’端添加腺苷酸和/或胞苷酸的酶可以是本领域周知的各种聚合酶和末端转移酶，所述聚合酶包括各种能为单链RNA分子3’端添加ATP、UTP、CTP和/或GTP的RNA聚合酶和DNA聚合酶。示例性的聚合酶包括但不限于poly(A)聚合酶(如大肠杆菌poly(A)聚合酶)、poly(U)聚合酶和DNA聚合酶θ(DNA polymerase theta，POLQ)。示例性的末端转移酶包括各种RNA末端转移酶和DNA末端转移酶(terminal deoxynucleotidyl transferase，TdT)。可根据需要使用一种或多种合适的酶实施所述聚合反应。在一些实施方案中，采用poly(A)聚合酶如大肠杆菌poly(A)聚 合酶实施本文所述的聚合反应。
聚合反应的反应体系可含有起始RNA分子、修饰的ATP和/或修饰的CTP、任选的未修饰ATP、反应的缓冲液、RNA酶抑制剂和酶等成分。反应可在常规的反应条件下进行。在一些实施方案中，反应体系中不含有未修饰ATP。在一些实施方案中，反应体系中含有修饰的ATP和/或修饰的CTP与未修饰的ATP，其中，修饰的ATP和/或修饰的CTP与未修饰ATP的比例在1∶19到19∶1的范围内，如1∶9到9∶1、2∶8到8∶2或4∶6到6∶4。在一些实施方案中，修饰的ATP和/或修饰的CTP与未修饰ATP的比例约为5∶5。
本文中，起始RNA分子可采用本领域周知的方法制备得到。例如，作为起始RNA分子的起始mRNA分子的制备方法可包括：使用poly(A)聚合酶加尾；使用带有poly(A)的DNA引物进行PCR扩增，得到含有poly(A)的DNA模板，通过体外转录(IVT)制备含poly(A)的RNA；带有poly(A)的DNA模板也可以通过将poly(A)序列直接插入到质粒载体中获得。质粒载体是目前使用最为广泛的主流方法，质粒载体在用作体外转录模板前先进行线性化，需要在质粒DNA模板的poly(A)序列后引入限制性内切酶切割位点。
在本发明的一些实施方案中，使用DNA模板通过体外转录制备得到起始mRNA分子。
在一些特别优选的实施方案中，起始mRNA分子采用具有以下结构的DNA模板制备得到：该DNA模板从5’端到3’端含有依次连接的启动子序列、5’非翻译区、Kozak序列、开放阅读框、3’非翻译区、poly(A)尾以及核酶序列。所述5’非翻译区、Kozak序列、开放阅读框和3’非翻译区如前文任一实施方案所述。
本文中，核酶指具有催化活性的RNA分子，其化学本质是核糖核酸(RNA)，却具有酶的催化功能。在一些实施方案中，本发明的核酶是具有自剪切功能的核酶，包括但不限于HDV核酶、发夹状核酶和锤头状核酶。其它可以自催化切割或通过蛋白因子介导切割的核苷酸序列也可用于本发明。在一些实施方案中，本发明使用其核苷酸序列如SEQ ID NO：1第113-197位所示的HDV核酶。
本文中，所述启动子可以是本领域制备mRNA时所使用的各类启动子。 启动子包括真核表达系统和原核表达系统的启动子。示例性的真核表达系统的启动子包括但不限于CMV启动子、EF1a启动子、SV40启动子、PGK1启动子、CAG启动子、TRE启动子、GDS启动子、Ac5启动子、ADH1启动子、Ubc启动子、UAS启动子、GAL1启动子、TEF1启动子、H1启动子和U6启动子等。常用的原核表达系统启动子包括T7启动子、T7lac启动子、Sp6启动子、araBAD启动子、trp启动子、lac启动子、Ptac启动子和pL启动子等。
该DNA模板可以载体的形式提供。示例性的载体包括但不限于pUC19，pBR322，pBluescript等。在本发明的载体中，在该DNA模板的两端设有合适的酶切位点。可采用本领域周知的方法构建本发明的载体。在一些实施方案中，本发明的载体是质粒载体，能在宿主细胞中自主复制。
应理解，当使用含核酶序列的DNA模板时，合成得到的RNA分子3’端的核酶进行自剪切，将在RNA分子的3’端形成腺苷酸-2’，3’-环磷酸。此时，可使用T4多聚核苷酸激酶进行处理，然后进行下一步的连接反应或聚合反应。
在一些实施方案中，RNA分子中，延长poly(A)尾后所得到的新的全长poly(A)尾的长度可为31-500个腺苷酸，其含有多个未修饰的腺苷酸和一个或多个修饰的腺苷酸。结构上，可将RNA分子已有的poly(A)尾视作腺苷酸序列1，其长度通常为30-250个连续的腺苷酸，在该poly(A)尾以外延长出来的poly(A)部分视作腺苷酸序列2，其长度通常为1-250个核苷酸。腺苷酸序列1可由未修饰的腺苷酸组成，或者含有未修饰的腺苷酸和修饰的腺苷酸。腺苷酸序列2含有至少一个修饰的腺苷酸。
在一些实施方案中，所述腺苷酸序列1的长度至少为50个核苷酸，优选至少60个核苷酸。在一些实施方案中，所述腺苷酸序列1的长度为50-200个核苷酸。在一些实施方案中，所述腺苷酸序列的长度为60-200个核苷酸。在一些实施方案中，所述腺苷酸序列1的长度为60-150个核苷酸。
在一些实施方案中，该腺苷酸序列1含有修饰的腺苷酸时，硫代腺苷不是唯一的修饰腺苷酸。在一些实施方案中，该腺苷酸序列1中不含有硫代腺苷修饰。
在一些实施方案中，所述腺苷酸序列2含修饰腺苷酸和/或修饰胞苷酸与 任选的未修饰腺苷酸。在一些实施方案中，该腺苷酸序列2中至少有2个、至少3个、至少4个、至少5个、至少10个核苷酸是修饰的腺苷酸和/或修饰胞苷酸。在一些实施方案中，该腺苷酸序列2中至少1％的核苷酸是修饰的腺苷酸和/或修饰胞苷酸。在一些实施方案中，该腺苷酸序列2中至少5％的核苷酸是修饰的腺苷酸和/或修饰胞苷酸。在一些实施方案中，该腺苷酸序列2中至少10％的核苷酸是修饰的腺苷酸和/或修饰胞苷酸。在一些实施方案中，该腺苷酸序列2中1-100％的核苷酸是修饰的腺苷酸和/或修饰胞苷酸，如1-100％、10-100％、20-100％、30-100％、40-100％、50-100％等，或者可为1-60％、5-60％、10-60％、15-60％或20-50％等。在一些实施方案中，该腺苷酸序列2中，修饰的腺苷酸中至少包括PS修饰的腺苷酸。优选地，修饰的腺苷酸中至少1％以上、优选至少5％以上、优选至少10％以上、更优选至少30％以上、优选至少50％以上的修饰腺苷酸为PS修饰的腺苷酸。在一些实施方案中，该腺苷酸序列2中，所有的修饰腺苷酸都是PS修饰的腺苷酸。在一些实施方案中，该腺苷酸序列2中，所含的修饰的胞苷酸是LNA修饰的胞苷酸。
在一些实施方案中，该腺苷酸序列2的长度为5个以上的核苷酸。在一些实施方案中，该腺苷酸序列2的长度为10个以上的核苷酸。在一些实施方案中，该腺苷酸序列2的长度为10-250个核苷酸。在一些实施方案中，该腺苷酸序列2的长度为20-200个核苷酸。在一些实施方案中，该腺苷酸序列2的长度为30-200个核苷酸。在一些实施方案中，该腺苷酸序列2的长度为150个以下的核苷酸。在一些实施方案中，该腺苷酸序列2的长度为5-150个核苷酸、10-150个核苷酸、20-150个核苷酸、30-150个核苷酸、20-100个核苷酸、20-50个核苷酸等。
在一些实施方案中，所述腺苷酸序列1由未修饰的腺苷酸组成，所述腺苷酸序列2含有修饰的腺苷酸和/或修饰胞苷酸与任选的未修饰的腺苷酸，且该腺苷酸序列2与腺苷酸序列1的3’端连接的第1个核苷酸是修饰的腺苷酸。
在一些实施方案中，起始RNA分子具有5’帽结构，因此制备得到的RNA分子还包含至少一个5’帽结构。示例性的5’帽结构包括但不限于Cap0(m7GpppXpYp)、Cap1(m7GpppXmpYp)、Cap2(m7GpppXmpYmp)；抗 反转帽子类似物(anti-reverse cap analog，ARCA)、β-S-ARCA、5’-LNA-Cap、1，5’-LNA-Cap以及N7-苄基二核苷四磷酸帽类似物。可采用本领域周知的技术对该RNA分子进行加帽。例如，对于mRNA分子，在制备得到mRNA分子后对其进行5’加帽和3’加poly(A)尾处理，得到本文所述的起始RNA分子，然后再通过本文所述的方法延长该poly(A)尾。
在一些实施方案中，本发明提供一种RNA分子。该RNA分子具有提升的胞内翻译效率和/或稳定性。该RNA分子具有poly(A)尾，其poly(A)尾的长度为31-500个腺苷酸，其含有多个未修饰的腺苷酸以及一个或多个修饰的腺苷酸和/或一个或多个修饰的胞苷酸；该poly(A)尾具有长度为30-250个连续核苷酸的腺苷酸序列1和长度为1-250个连续核苷酸的腺苷酸序列2。该腺苷酸序列1可由未修饰的腺苷酸组成，或者含有未修饰的腺苷酸和修饰的腺苷酸。该腺苷酸序列2含有至少一个修饰的腺苷酸和/或至少一个修饰的胞苷酸。在一些实施方案中，所述腺苷酸序列1和2如前文任一实施方案所述。尤其是，在一些实施方案中，该腺苷酸序列1含有修饰的腺苷酸时，硫代腺苷不是唯一的修饰腺苷酸；在一些实施方案中，该腺苷酸序列1中不含有硫代腺苷修饰；在另外一些实施方案中，所述腺苷酸序列1由未修饰的腺苷酸组成，所述腺苷酸序列2含有修饰的腺苷酸和/或修饰的胞苷酸与任选的未修饰的腺苷酸，且该腺苷酸序列2与腺苷酸序列1的3’端连接的第1个核苷酸是修饰的腺苷酸。在一些实施方案中，该腺苷酸序列1长50-200个、优选60-200个、更优选60-150个核苷酸，该腺苷酸序列2长1-250个、如5-200个、或10-200个、或10-150个核苷酸，且该腺苷酸序列2中1-100％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸，如5-100％、10-100％、20-100％、30-100％、40-100％、50-100％等，或者可为5-60％、10-60％、15-60％或20-50％等。
该RNA分子中除poly(A)尾外的其余部分如前文任一实施方案所述。在一些有些的实施方案中，该RNA分子是mRNA分子。优选地，该mRNA分子具有前文任一实施方案所述的生物学功能和结构。
在一些实施方案中，该RNA分子是采用本文任一实施方案所述的RNA分子制备方法制备得到。
在一些实施方案中，本申请提供一种poly(A)分子。该poly(A)分子可独立存在或作为RNA分子的一部分。当作为RNA分子的一部分时，其存在有助于提高该RNA分子在胞内的翻译效率和/或稳定性。该poly(A)分子的长度为31-500个腺苷酸，其含有多个未修饰的腺苷酸以及一个或多个修饰的腺苷酸和/或一个或多个修饰的胞苷酸；该poly(A)尾具有长度为30-250个连续核苷酸的腺苷酸序列1和长度为1-250个连续核苷酸的腺苷酸序列2。该腺苷酸序列1可由未修饰的腺苷酸组成，或者含有未修饰的腺苷酸和修饰的腺苷酸。该腺苷酸序列2含有至少一个修饰的腺苷酸和/或至少一个修饰的胞苷酸。在一些实施方案中，所述腺苷酸序列1和2如前文任一实施方案所述。尤其是，在一些实施方案中，该腺苷酸序列1含有修饰的腺苷酸时，硫代腺苷不是唯一的修饰腺苷酸；在一些实施方案中，该腺苷酸序列1中不含有硫代腺苷修饰；在另外一些实施方案中，所述腺苷酸序列1由未修饰的腺苷酸组成，所述腺苷酸序列2含有修饰的腺苷酸和/或修饰的胞苷酸与任选的未修饰的腺苷酸，且该腺苷酸序列2与腺苷酸序列1的3’端连接的第1个核苷酸是修饰的腺苷酸。在一些实施方案中，该腺苷酸序列1长50-200个、优选60-200个、更优选60-150个核苷酸，该腺苷酸序列2长1-250个、如5-200个、或10-200个、或10-150个核苷酸，且该腺苷酸序列2中1-100％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸，如5-100％、10-100％、20-100％、30-100％、40-100％、50-100％等，或者可为5-60％、10-60％、15-60％或20-50％等。
在一些实施方案中，本发明提供一种药物组合物，该药物组合物含有本发明任一实施方案所述的RNA分子和药学上可接受的载体。在一些实施方案中，所述药物组合物是疫苗，即核酸疫苗。在一些实施方案中，所述疫苗是肿瘤疫苗。在一些实施方案中，所述疫苗是预防性疫苗和治疗性疫苗。预防性疫苗中mRNA分子编码抗原多肽。治疗性疫苗中，mRNA分子编码治疗性多肽，包括肿瘤相关性抗原多肽(TAA)和肿瘤特异性抗原多肽(TSA)。本文中，药学上可接受的载体可以是本领域周知的用于递送mRNA分子的药学上可接受的载体、赋形剂等。在一些实施方案中，该药物组合物是含有所述mRNA分子和佐剂的疫苗。佐剂可以是mRNA疫苗常用佐剂。
在一些实施方案中，本发明提供本文任一实施方案所述的修饰的ATP、修饰的CTP、修饰ATP与未修饰ATP的混合物、修饰CTP与未修饰ATP的混合物、修饰ATP和修饰CTP和未修饰ATP的混合物、或由修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子在制备胞内翻译效率和/或稳定性提高的RNA分子中的应用。优选地，用于所述制备的起始RNA分子已具有poly(A)尾。在一些实施方案中，本发明提供修饰的腺苷酸和/或修饰胞苷酸或由修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子在提高已有poly(A)尾的RNA分子的细胞内翻译效率和/或稳定性中的应用。在一些实施方案中，所述poly(A)尾为本申请任一实施方案所述的腺苷酸序列1。在一些实施方案中，所述修饰ATP与未修饰ATP的混合物、修饰CTP与未修饰ATP的混合物、修饰ATP和修饰CTP和未修饰ATP的混合物中，修饰的ATP和/或修饰的CTP和未修饰ATP的比例在1∶19到19∶1的范围内，如在1∶9到9∶1、2∶8到8∶2或4∶6到6∶4的范围内，或者可为5∶5。在一些实施方案中，所述由修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子为本文任一实施方案所述的腺苷酸序列2。在一些实施方案中，所述由修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子不含有未修饰腺苷酸，或含有时，修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸的比例在1∶19到19∶1的范围内，如在1∶9到9∶1、2∶8到8∶2或4∶6到6∶4的范围内，或者可为5∶5。在一些实施方案中，所述修饰包括针对磷酸基的修饰和针对糖基的修饰，包括但不限于以硫代磷酸(phosphorothioate，PS)修饰、硼代磷酸(boranophosphate，PB)修饰、甲基代磷酸(methylphosphonate，MP)修饰为代表的磷酸基修饰和以锁核酸(locked nucleic acid，LNA)修饰、2’甲氧基(2’-O-Methyl，2’OMe)修饰、2’氟代(2’-Fluoro，2’F)修饰为代表的糖基修饰。在一些实施方案中，所述修饰ATP为PS修饰的ATP、LNA修饰的ATP、PB修饰的ATP、MP修饰的ATP、PS修饰的ATP与2’OMe修饰的ATP的混合物、PS修饰的ATP与2’F修饰的ATP的混合物、PS修饰的ATP与PB修饰的ATP的混合物、或PS修饰的ATP与MP修饰的ATP的混合物。修饰的CTP的例子包括LNA修饰的CTP。
本发明还提供一种试剂盒，其含有本文任一实施方案所述的修饰ATP和/或修饰CTP与未修饰ATP的混合物，或含有本文任一实施方案所述的由修饰腺苷酸和/或修饰胞苷酸和任选的未修饰腺苷酸组成的poly(A)分子。本发明的试剂盒可用于通过聚合反应或连接反应(尤其是本文任一实施方案所述的聚合反应或连接)在RNA分子已具有的poly(A)尾的3’端加入含修饰腺苷酸和/或修饰胞苷酸与任选的未修饰腺苷酸的poly(A)序列。因此，本发明的试剂盒中还可含有用于实施聚合反应或连接反应的试剂。例如，用于实施连接反应的试剂包括但不限于ATP/CTP、反应缓冲液和连接酶等；用于实施聚合反应的试剂包括酶(如前文所述的聚合酶和/或末端转移酶，例如poly(A)聚合酶，尤其是E.coli poly(A)聚合酶)、RNA酶抑制剂和反应缓冲液等。在一些实施方案中，本发明的试剂盒还包括制备待进行所述连接反应或聚合反应的RNA分子的试剂。示例性的制备mRNA分子的方法包括：使用poly(A)聚合酶加尾；使用带有poly(A)的DNA引物进行PCR扩增，得到含有poly(A)的DNA模板，通过体外转录(IVT)制备含poly(A)的RNA；以及将poly(A)序列直接插入到质粒载体中，得到含有poly(A)的DNA模板，通过体外转录(IVT)制备含poly(A)的RNA。RNA分子的制备可以包括或不包括5’加帽反应。因此，本发明的试剂盒中可含有实施这些方法/步骤所需的试剂。
本发明不同于在整条mRNA中或者在整个poly(A)尾中添加修饰核苷酸的方法，而是在已有的mRNA poly(A)尾的3’端再添加带有修饰的多聚核苷酸。这种仅针对mRNA 3’端poly(A)尾的后部额外添加修饰核苷酸的方法，显著提升了mRNA的蛋白质表达效率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例一
材料与方法
1.体外转录模板的制备
构建萤火虫荧光素酶报告基因(Fluc)及海肾荧光素酶(Rluc)报告基因的质粒载体，经酶切反应和纯化获得用于PCR扩增的DNA模板。使用带有poly(A)的DNA引物进行PCR扩增，可得到含有poly(A)的DNA片段作为体外转录RNA的模板。在PCR扩增反应中，使用终浓度为0.4μM的正向引物(forward primer)和反向引物(reverse primer)，以及1ng/ul DNA模板、0.2mM dNTP、1.5mM MgSO4、1×KOD反应缓冲液、KOD酶(高保真DNA聚合酶)。根据说明书设置反应条件(三步法)，95℃ 3分钟后，反应30个循环，最后延伸68℃ 5分钟。循环条件为：变性98℃ 10秒，退火56℃ 30秒，延伸68℃ 3分钟。反应结束后使用DNA纯化试剂盒对产物进行纯化。
2.mRNA的合成
使用含有poly(A)的DNA模板，通过体外转录(IVT)制备含poly(A)的mRNA。在体外转录反应中，使用终浓度为1.6mM的帽子类似物Cap1和终浓度为2mM的ATP、UTP、CTP、GTP，以及20ng/ul DNA模板、1×T7反应缓冲液、T7 RNA聚合酶、RNA酶抑制剂，在37℃反应4小时后终止反应，加入DNase I在37℃反应15分钟以去除DNA模板，再使用MEGAclear转录纯化试剂盒(Thermo Fisher)对产物进行纯化。通过体外转录反应制备含有修饰核苷酸的mRNA时，可将体外转录反应体系中的ATP/UTP/CTP/GTP部分替换为修饰的ATP/UTP/CTP/GTP。例如将部分UTP替换为假尿嘧啶核苷修饰的UTP，将部分ATP替换为2’甲氧基腺苷或2’氟代腺苷修饰的ATP。
3.RNA连接反应
使用T4RNA连接酶1进行mRNA与RNA oligonucleotides(简称为RNA oligo)的连接反应。为了保证连接效率，RNA oligo均为5’P、3’ddC修饰。在20ul的连接反应中，使用2pmol mRNA和50pmol RNA oligo，以及1mM ATP、 20％PEG8000、10％DMSO、1×T4连接酶反应缓冲液、T4连接酶I、RNA酶抑制剂，在25℃反应2小时后终止反应，使用MEGAclear转录纯化试剂盒(Thermo Fisher)对产物进行纯化。
4.E.coli poly(A)聚合酶加尾反应
使用E.coli poly(A)聚合酶，以ATP及修饰ATP为底物，为RNA 3’端添加修饰核苷酸。在20ul的加尾反应中，使用10ug RNA和终浓度为1mM的ATP及修饰ATP，以及1×E.coli poly(A)聚合酶反应缓冲液、E.coli poly(A)聚合酶、RNA酶抑制剂，在37℃反应15-60分钟后终止反应，使用MEGAclear转录纯化试剂盒(Thermo Fisher)对产物进行纯化。
5.mRNA的琼脂糖凝胶电泳
配制1.5％的琼脂糖凝胶，使用E.coli poly(A)聚合酶的加尾反应后获得的RNA，进行电压160V电泳。
6.mRNA转染细胞及报告基因实验
分别使用不同的萤火虫荧光素酶报告基因(firefly luciferase，简称Fluc)的mRNA转染细胞，并同时转染等量海肾荧光素酶(renilla luciferase，简称Rluc)报告基因的mRNA作为参照。以24孔板为例：当HEK293T细胞密度约为40％时，将0.22μl lipofectamine2000稀释于50μl Opti-MEM中，室温静置5分钟后，加入预先稀释于50μl Opti-MEM的100ng Fluc mRNA和10ng Rluc mRNA中，混匀后再室温静置20分钟，最后小心加入细胞中，培养24、36、48小时后，分别用1×PBS漂洗细胞，再用适量裂解缓冲液(100mM Potassium Phosphate[pH 7.8]，0.2％(v/v)Triton X-100)裂解细胞，在室温10000rpm离心1分钟，取适量上清，加入相应体积的双荧光素酶报告基因检测试剂盒里提供的荧光素酶反应底物，用多功能微孔板检测仪记录荧光值。将Fluc荧光值除以Rluc荧光值，再乘以该时间点各样品Rluc荧光值的平均值，即为经Rluc值标准化后的Fluc值(Normalized Fluc)，作为mRNA在不同时间点的蛋白质 表达效率的参考值。
实验结果
1.为了测试核苷酸修饰的作用，我们首先设计了含有硫代磷酸(phosphorothioate，PS)修饰的A20-PS RNA oligo(仅含有3个硫代磷酸修饰的A)、含有锁核酸(locked nucleic acid，LNA)修饰的A20-LNA RNA oligo(仅含有2个锁核酸修饰的A)、含有锁核酸(LNA)修饰的A20-LNAC RNA oligo(仅含有2个锁核酸修饰的C)和不含修饰的A20 RNA oligo(图2，a-b)。我们制备了poly(A)长度为60个A(A60)的萤火虫荧光素酶(Fluc)报告基因的mRNA，将不同RNA oligo分别连接到Fluc-A60 mRNA的3’端(图2，b)，并将连接后的mRNA分别转染至HEK293T细胞中。我们发现，尽管上述mRNA的poly(A)3’末端只引入了3个硫代磷酸修饰，其在细胞内的蛋白质表达效率显著高于无修饰的对照组mRNA(图2，c)，说明mRNA poly(A)结构中的硫代磷酸修饰具有提升mRNA蛋白质表达效率的潜力。此外，在poly(A)3’末端引入2个锁核酸修饰的A或C后，mRNA在细胞内的蛋白质表达效率也有所提升(图2，d和e)。
2.硫代磷酸修饰的三磷酸腺苷又称为ATPαS(Adenosine-5′-O-(1-Thiotriphosphate)，1-Thio-ATP)，结构如图3(a)所示。为了选择最合适的ATPαS∶ATP混合比例，我们首先将反应体系中ATP与ATPαS的总终浓度设定为1mM，设置的ATPαS∶ATP比例分别为0∶10、1∶9、2∶8、3∶7、4∶6、5∶5、6∶4、7∶3、8∶2、9∶1、10∶0、0∶0。我们给70bp的3’端为30个未修饰A的短RNA进行上述加尾反应(图3，b)。经30min、60min的加尾反应得到的RNA，使用1.5％琼脂糖凝胶进行电泳，通过RNA条带的大小变化，来判断加尾的长度。在ATPαS∶ATP比例为8∶2、9∶1、10∶0时，与0∶0相比，RNA条带没有明显迁移；在ATPαS∶ATP比例为0∶10、1∶9、2∶8、3∶7、4∶6、5∶5、6∶4时，RNA出现明显迁移，其中从ATPαS∶ATP比例为5∶5时，RNA条带较为集中，代表RNA的均一性较好(图3，c)。
3.我们将E.coli poly(A)聚合酶反应体系中ATP与ATPαS的总终浓度分别设定为0.1mM、0.3mM、1mM，并将ATPαS∶ATP比例分别设定为0∶10、2∶8、5∶5、8∶2、10∶0、0∶0，为Fluc-A60 mRNA进行加尾反应(图4，a)。由于Fluc-A60 mRNA片段较大(约2kb)，所以加尾200个腺苷酸以内时难以观察到mRNA条带的迁移(图4，b)。我们将mRNA转染至293T细胞中，检测其蛋白质表达效率。相比于仅加ATP的对照组(ATPαS∶ATP＝0∶10)以及未进行反应的对照组(control)，在这次实验中，含有硫代磷酸修饰的mRNA的蛋白质表达效率都更高(图4，c)；并且在反应总终浓度为1mM、ATPαS∶ATP比例为5∶5时，mRNA的蛋白质表达效率最高，远高于对照组，再次证明了mRNA poly(A)结构中的硫代磷酸修饰在提升mRNA蛋白质表达效率方面的作用。
4.我们以上实验发现，使用ATPαS在已带有一定长度poly(A)尾的mRNA 3’端添加硫代磷酸修饰能提升mRNA的蛋白质表达效率。为研究mRNA poly(A)结构中的硫代磷酸修饰的作用是否会随其位置改变而发生变化，我们分别针对含poly(A)长度为A60的Fluc mRNA、不含poly(A)的Fluc mRNA，使用ATP以及ATP+ATPαS(即ATPαS∶ATP＝5∶5)进行加尾反应(图4，a)，反应体系中ATP与ATPαS的总终浓度设定为1mM。结果发现，无poly(A)尾的A0 mRNA的蛋白质表达效率极低；相比于未进行加尾反应的A60 mRNA，仅使用ATP进行加尾反应的A0 ATP mRNA、A60 ATP mRNA的蛋白质表达效率略有提升；而在使用ATP和ATPαS各50％进行加尾反应所得到的mRNA中，A0 ATP+ATPαS mRNA的蛋白质表达效率提升幅度较小，A60 ATP+ATPαS mRNA的蛋白质表达效率提升幅度更大(图4，d)。这说明了mRNA poly(A)结构中的硫代磷酸修饰的位置，确实会显著影响mRNA的表达效率。其中在mRNA的无修饰poly(A)尾的3’端继续添加硫代磷酸修饰腺苷酸能大幅提升mRNA在细胞内的蛋白质表达效率。
5.在E.coli poly(A)聚合酶反应中，除了硫代磷酸修饰核苷酸(ATPαS)， 我们还测试了2’甲氧基修饰核苷酸(2’OMe-ATP)与2’氟代修饰核苷酸(2’F-dATP)、硼代磷酸修饰核苷酸(ATPαBH3)与甲基代磷酸修饰核苷酸(ATPαCH3)(图5，a)。将ATP与修饰ATP总终浓度设定为1mM，Fluc-A60 mRNA分别使用ATP、50％ATP+50％ATPαS、50％ATP+25％ATPαS+25％2’OMe-ATP、50％ATP+25％ATPαS+25％2’F-dATP进行加尾反应；此外，Fluc-A60 mRNA分别使用ATP、50％ATP+50％ATPαS、50％ATP+50％ATPαBH3、50％ATP+50％ATPαCH3、50％ATP+25％ATPαS+25％ATPαBH3、50％ATP+25％ATPαS+25％ATPαCH3进行加尾反应(图5，b)。结果发现，在mRNA 3’端poly(A)尾的3’端，通过E.coli poly(A)聚合酶添加ATPαS与2’OMe-ATP或2’F-dATP的混合物能显著提升mRNA在细胞内表达蛋白质的效率(图5，c)。在mRNA 3’端poly(A)尾的3’端，通过E.coli poly(A)聚合酶添加ATPαBH3、ATPαCH3以及ATPαS与ATPαBH3或ATPαCH3的混合物能显著提升mRNA在细胞内表达蛋白质的效率(图5，d)。
6.综上，本发明在起始RNA分子3’端已有的poly(A)尾3’端后引入修饰的腺苷酸，形成延长的poly(A)序列(图1)，可以显著提高RNA分子在细胞内的稳定性和/或翻译效率，实现使用更少量的mRNA在细胞内表达更多的蛋白质，有效地降低mRNA使用剂量和生产成本。
为了探索以上在已有的poly(A)尾3’端后引入修饰的腺苷酸的方法是否适用于整条带有假尿嘧啶核苷修饰的起始mRNA分子，我们在体外转录反应体系中将UTP替换为50％假尿嘧啶核苷修饰的UTP+50％UTP，得到带有假尿嘧啶核苷修饰的Fluc-A60 mRNA(图6，a)。结果发现，在带有假尿嘧啶核苷修饰的mRNA的poly(A)尾的3’端，通过E.coli poly(A)聚合酶添加硫代磷酸修饰，仍然能显著提升mRNA在细胞内表达蛋白质的效率(图6，b)。类似地，为了探索以上在已有的poly(A)尾3’端后引入修饰的腺苷酸的修饰方法是否适用于整条带有2’甲氧基腺苷或2’氟代腺苷修饰的起始mRNA分子，我们在体外转录反应体系中将ATP替换为10％2’甲氧基修饰的ATP+90％ATP，得到 带有2’甲氧基腺苷修饰的mRNA；或者在体外转录反应体系中将ATP替换为10％2’氟代修饰的ATP+90％ATP，得到带有2’氟代腺苷修饰的mRNA(图6，c)。结果发现，在带有2’甲氧基腺苷或2’氟代腺苷修饰的mRNA的poly(A)尾的3’端，通过E.coli poly(A)聚合酶添加硫代磷酸修饰，仍然能显著提升mRNA在细胞内表达蛋白质的效率(图6，d-e)，降低mRNA使用剂量和生产成本。
实施例二
材料与方法
1.质粒构建
在金斯瑞公司人工合成2种含有poly(A)序列和HDV核酶序列的DNA片段，构建到pUC57-simple质粒上。片段名称分别为：XhoI-A100-RZ-BglII片段、XhoI-A100-NotIA-RZ-BglII片段。
XhoI-A100-RZ-BglII片段从5’端到3’端序列为(SEQ ID NO：1)：
其中下划横线所示序列为XhoI、BglII位点，其中下划波浪线所示序列为HDV核酶(简写RZ)序列。
XhoI-A100-NotIA-RZ-BglII片段从5’端到3’端序列为(SEQ ID NO：2)：
其中下划横线所示序列为XhoI、NotI、BglII位点，其中下划波浪线所示 序列为HDV核酶(简写RZ)序列，NotI和HDV核酶序列之间间隔一个A。
使用限制性内切酶XhoI和BglII将以上2个DNA片段从金斯瑞公司提供的pUC57-simple质粒上切下来，做DNA切胶回收，作为插入片段(Insert)；使用已构建好的含有T7启动子序列、5’非翻译区、Kozak序列、萤火虫荧光素酶报告基因(Fluc)的开放阅读框、3’非翻译区的质粒，使用限制性内切酶XhoI和BglII进行酶切，做DNA切胶回收得到Fluc载体片段。
再使用T4 DNA连接酶进行连接反应，分别将XhoI-A100-RZ-BglII片段、XhoI-A100-NotIA-RZ-BglII片段连接到Fluc载体片段上，用感受态细胞DH5α和Stbl3进行转化，挑选单克隆进行sanger测序，确认序列正确，分别得到质粒Fluc-A100RZ和Fluc-A100NotIARZ。
2.DNA模板的线性化
用限制性内切酶将以上质粒中所需的用于体外转录的DNA模板切下，做DNA切胶回收，得到6种DNA模板。
(1)用限制性内切酶EcoRI和BglII切质粒Fluc-A100RZ，做DNA切胶回收得到模板Fluc-A100-RZ-DH5α和Fluc-A100-RZ-Stbl3。
(2)用限制性内切酶EcoRI和BglII切质粒Fluc-A100NotIARZ，做DNA切胶回收得到模板Fluc-A100-NotIA-RZ-DH5α和Fluc-A100-NotIA-RZ-Stbl3。
(3)用限制性内切酶EcoRI和NotI切质粒Fluc-A100NotIARZ，做DNA切胶回收得到模板Fluc-A100-NotIcut-DH5α和Fluc-A100-NotIcut-Stbl3。
3.体外转录得到mRNA
使用以上线性化的DNA模板作为体外转录的模板，用T7 RNA聚合酶进行体外转录反应，得到相应的mRNA分子。在体外转录反应中，使用终浓度为1.6mM的帽子类似物和终浓度为2mM的ATP、UTP、CTP，以及终浓度为0.4mM的GTP、20ng/ul DNA模板、1x T7反应缓冲液、T7 RNA聚合酶、RNA酶抑制剂，在37℃反应4小时后终止反应，加入DNase I在37℃反应15分钟以去除DNA模板，再使用MEGAclear转录纯化试剂盒(Thermo Fisher)对产 物进行纯化。如果使用含核酶序列的DNA模板，合成得到的RNA 3’端的核酶进行自剪切后将在RNA的3’端形成腺苷酸-2’，3’-环磷酸，此时可使用T4多聚核苷酸激酶进行处理后进行下一步的连接反应或聚合反应。
4.mRNA的PAGE变性胶电泳
配制6％PAGE、8M尿素的变性胶，使用纯化前的RZ-mRNA、NotIA-RZ-mRNA、NotIcut-mRNA进行200V 25min电泳。
5.mRNA转染细胞及报告基因实验
分别使用不同的萤火虫荧光素酶报告基因(firefly luciferase，简称Fluc)的mRNA转染细胞，并同时转染等量海肾荧光素酶(renilla luciferase，简称Rluc)报告基因的mRNA作为参照。以24孔板为例：当HEK293T细胞密度约为40％时，将0.22μl lipofectamine2000稀释于50μl Opti-MEM中，室温静置5分钟后，加入预先稀释于50μl Opti-MEM的100ng Fluc mRNA和10ng Rluc mRNA中，混匀后再室温静置20分钟，最后小心加入细胞中，培养24小时后，用1×PBS漂洗细胞，再用适量裂解缓冲液(100mM磷酸钾[pH 7.8]，0.2％(v/v)Triton X-100)裂解细胞，在室温10000rpm离心1分钟，取适量上清，加入相应体积的双荧光素酶报告基因检测试剂盒里提供的荧光素酶反应底物，用多功能微孔板检测仪记录荧光值。将萤火虫荧光素酶荧光值除以海肾荧光素酶荧光值(Fluc/Rluc比值)即为报告基因的表达情况，作为mRNA的蛋白质表达效率的参考值。
实验结果
本发明在质粒载体的poly(A)序列3’端引入了一个改造过的丁型肝炎病毒核酶(HDV RZ)序列(图7，a)，在体外转录反应生成mRNA分子后，3’端的核酶进行自剪切，形成具有固定长度的poly(A)尾，并且防止了poly(A)3’端的非A碱基残留(图7，b)。
为了比较新型体外转录模板与传统体外转录模板，我们构建了不同的萤火 虫荧光素酶(firefly luciferase，简称Fluc)报告基因质粒载体，制备相应的体外转录模板和mRNA，包括：(1)构建poly(A)序列3’端紧连着HDV核酶序列的质粒载体，记为Fluc-A100-RZ质粒；用限制性内切酶EcoRI和BglII将其线性化，得到DNA模板用于体外转录，生成poly(A)3’端含核酶、不含非A碱基的mRNA，命名为RZ mRNA(图8，a)。(2)构建poly(A)序列与HDV核酶序列之间含NotI酶切位点和1个A碱基的质粒载体，记为Fluc-A100-NotIA-RZ质粒；用限制性内切酶EcoRI和BglII将其线性化，得到DNA模板用于体外转录；生成poly(A)3’端含核酶、含非A碱基的mRNA，命名为NotIA-RZ mRNA(图8，b)。(3)使用第(2)点中同样的Fluc-A100-NotIA-RZ质粒，但是用限制性内切酶EcoRI和NotI将其线性化，得到DNA模板用于体外转录；生成poly(A)3’端不含核酶、含非A碱基的mRNA，命名为NotIcut mRNA(图8，c)。
构建获得的Fluc-A100-RZ质粒和Fluc-A100-NotIA-RZ质粒分别转入菌株DH5a和Stbl3感受态细胞进行质粒扩增，提取的质粒经体外转录合成的mRNA分别标记为：RZ-D、RZ-S；NotIA-RZ-D、NotIA-RZ-S；NotIcut-D、NotIcut-S，其中后缀-D代表来自DH5a菌株的克隆，后缀-S代表来自Stbl3菌株的克隆。使用6％PAGE变性胶进行电泳，发现RZ、NotIA-RZ mRNA的样品中，在100bp左右位置出现RNA条带，即核酶自剪切条带(图9，a)，mRNA经过纯化后即可完全去除该核酶分子，不会对后续的反应和细胞实验造成任何影响。
我们将这6种Fluc mRNA分别转染至HEK293T细胞中，24h后检测其蛋白质表达效率。结果发现，poly(A)3’端不含非A碱基的RZ mRNA的蛋白质表达效率显著高于poly(A)3’端含若干个非A碱基的NotIA-RZ(RZ核酶切割后仍然带有若干个非A碱基)、NotIcut mRNA(不带有RZ核酶)的(图9，b-d)，说明去除poly(A)3’端非A碱基对于提高mRNA在细胞内表达效率的重要性。
以上实验结果说明在mRNA的poly(A)3’端添加自切割核酶，能形成准确均一的、不带有其他多余核苷酸的poly(A)尾，从而提高mRNA的蛋白质翻译效率。

Claims (10)

  1. RNA分子的制备方法，其特征在于，所述方法包括在起始RNA分子的poly(A)尾的3’端添加由修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸组成的修饰poly(A)序列的步骤。
  2. 如权利要求1所述的方法，其特征在于，所述方法具有以下一项或多项特征：
    (1)所述方法用于制备翻译效率和/或稳定性提高的RNA分子；
    (2)所述poly(A)尾为长度为30-250个、优选50-200个、更优选60-200个、更优选60-150个连续的含有未修饰腺苷酸并任选含有修饰腺苷酸的腺苷酸序列1，所述修饰poly(A)序列为长度为1-250个、优选10-250个、更优选20-200个、更优选30-200个核苷酸的腺苷酸序列2；优选地，所述腺苷酸序列2中至少1％、优选5％、优选10-100％、更优选20-100％、更优选50-100％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸；
    (3)采用连接反应或聚合反应在所述起始RNA分子的所述poly(A)尾3’端添加所述修饰poly(A)序列；
    优选地，所述连接反应中，使用RNA连接酶将所述修饰poly(A)序列与所述poly(A)尾连接；优选地，所述修饰poly(A)序列的5’端具有5’P修饰，3’端具有选自以下的修饰：3’ddC、3’BHQ-1、3’BHQ-2、3’MGB、3’Dabcul、3’6-TAMRA、3’6-FAM、3’CY5、3’CY3、3’6-ROX、3’inverted dT、3’Biotin、3’Biotin-TEG、3’Amino modified C7、3’Phosphate、3’Digoxigenin、3’Thiol modifier C6 S-S、3’Thiol modifier C3 S-S、3’Spacer C3、3’Spacer C6、3’Spacer 9、3’Spacer 18、3’dSpacer、3’Cholesteryl-TEG和3’Ferrocene dT；
    优选地，所述聚合反应中，使用聚合酶或末端转移酶以修饰ATP及任选的未修饰ATP为底物，在起始RNA分子的所述poly(A)尾的3’端添加修饰腺苷酸和任选的未修饰腺苷酸；优选地，聚合反应的反应体系不含有未修饰ATP，或含有未修饰ATP时，修饰ATP和未修饰ATP的比例在1∶19到19∶1的范围内，如1∶9到9∶1或2∶8到8∶2；
    (4)所述起始RNA分子含有修饰的核苷酸；
    (5)所述方法还包括通过DNA模板制备具有所述poly(A)尾的起始RNA分子的步骤；优选地，所述DNA模板从5’端到3’端含有依次连接的启动子序列、5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和poly(A)尾序列；更优选地，所述DNA模板在该poly(A)尾之后还直接连接有核酶序列；优选地，所述核酶是具有自剪切功能的核酶，优选选自HDV核酶、发夹状核酶和锤头状核酶；
    (6)所述修饰腺苷酸和修饰胞苷酸中的修饰包括针对磷酸基的修饰和针对糖基的修饰；优选地，所述修饰选自硫代磷酸(PS)修饰、硼代磷酸(PB)修饰、甲基代磷酸(MP)修饰、锁核酸(LNA)修饰、2’甲氧基(2’OMe)修饰和2’氟代(2’F)修饰；优选地，所述修饰腺苷酸为PS修饰的腺苷酸、PB修饰的腺苷酸、MP修饰的腺苷酸、LNA修饰的腺苷酸、PS修饰的腺苷酸与2’OMe修饰的腺苷酸的混合物、PS修饰的腺苷酸与PB修饰的腺苷酸、PS修饰的腺苷酸与MP修饰的腺苷酸、或PS修饰的腺苷酸与2’F修饰的腺苷酸的混合物，所述修饰胞苷酸为LNA修饰的胞苷酸。
  3. 一种mRNA分子的制备方法，其特征在于，所述方法包括以下步骤：
    (1)使用DNA模板通过体外转录制备得到具有poly(A)尾的mRNA分子；和
    (2)采用连接反应或聚合反应在步骤(1)获得的mRNA分子的所述poly(A)尾的3’端添加修饰腺苷酸和/或修饰胞苷酸和任选的未修饰腺苷酸或含有所述修饰腺苷酸和/或修饰胞苷酸和任选的未修饰腺苷酸的修饰的poly(A)序列，从而制备得到所述mRNA分子；优选地，以修饰ATP及任选的未修饰ATP为底物，使用聚合酶或末端转移酶在步骤(1)获得的mRNA分子的所述poly(A)尾的3’端添加修饰腺苷酸和任选的未修饰腺苷酸，从而制备得到所述mRNA分子。
  4. 如权利要求3所述的方法，其特征在于：
    步骤(1)中，所述DNA模板从5’端到3’端含有依次连接的启动子序列、5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和poly(A)尾；更优选地， 所述DNA模板在该poly(A)尾之后还直接连接有核酶序列；优选地，所述核酶是具有自剪切功能的核酶，优选选自HDV核酶、发夹状核酶和锤头状核酶；和/或
    步骤(2)的反应体系中不含有未修饰ATP，或含有修饰ATP和/或修饰CTP与未修饰ATP时，其中，修饰ATP和/或修饰CTP和未修饰ATP的比例在1∶19到19∶1的范围内，如1∶9到9∶1或2∶8到8∶2；和/或
    所述连接反应中，使用RNA连接酶将含有所述修饰腺苷酸和/或修饰胞苷酸和任选的未修饰腺苷酸的修饰poly(A)序列与所述mRNA分子的所述poly(A)尾的3’端连接；优选地，所述修饰poly(A)序列的5’端具有5’P修饰，3’端具有选自以下的修饰：3’ddC、3’BHQ-1、3’BHQ-2、3’MGB、3’Dabcul、3’6-TAMRA、3’6-FAM、3’CY5、3’CY3、3’6-ROX、3’inverteddT、3’Biotin、3’Biotin-TEG、3’Amino modified C7、3’Phosphate、3’Digoxigenin、3’Thiol modifier C6 S-S、3’Thiol modifier C3 S-S、3’Spacer C3、3’Spacer C6、3’Spacer 9、3’Spacer 18、3’dSpacer、3’Cholesteryl-TEG和3’Ferrocene dT；和/或
    所述聚合反应中，反应体系不含有未修饰ATP，或含有未修饰ATP时，修饰ATP和未修饰ATP的比例在1∶19到19∶1的范围内，如1∶9到9∶1或2∶8到8∶2。
  5. 一种RNA分子，其特征在于，该RNA分子具有含有多个未修饰的腺苷酸和一个或多个修饰的腺苷酸和/或一个或多个修饰胞苷酸的poly(A)尾；其中，该poly(A)尾具有长度为30-250个、优选50-200个、更优选60-200个、更优选60-150个连续核苷酸的腺苷酸序列1和与该腺苷酸序列1的3’端连接的长度为1-250个、优选10-250个、更优选20-200个、更优选30-200个连续核苷酸的腺苷酸序列2；其中，该腺苷酸序列1由未修饰的腺苷酸组成，或者含有未修饰的腺苷酸和修饰的腺苷酸，该腺苷酸序列2含有至少一个修饰的腺苷酸和/或至少一个修饰的胞苷酸；其中，该腺苷酸序列1含有修饰的腺苷酸时，硫代腺苷不是唯一的修饰腺苷酸，优选地，该腺苷酸序列1中不含有硫代腺苷修饰。
  6. 如权利要求5所述的RNA分子，其特征在于，所述RNA分子具有以 下一项或多项特征：
    (1)所述RNA分子选自mRNA分子、长链非编码RNA或非编码小RNA前体；
    (2)该腺苷酸序列2中至少1％、优选至少5％、优选至少10％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸；优选地，该腺苷酸序列2中优选10-100％、更优选20-100％、更优选50-100％的核苷酸是修饰的腺苷酸和/或修饰的胞苷酸；
    (3)所述腺苷酸序列1由未修饰的腺苷酸组成，所述腺苷酸序列2含有修饰的腺苷酸和/或修饰的胞苷酸与任选的未修饰的腺苷酸，且该腺苷酸序列2与腺苷酸序列1的3’端连接的第1个核苷酸是修饰的腺苷酸或修饰的胞苷酸；
    (4)所述修饰腺苷酸和修饰胞苷酸中的修饰包括针对磷酸基的修饰和针对糖基的修饰；优选地，所述修饰选自硫代磷酸(PS)修饰、硼代磷酸(PB)修饰、甲基代磷酸(MP)修饰、锁核酸(LNA)修饰、2’甲氧基(2’OMe)修饰和2’氟代(2’F)修饰；优选地，所述修饰腺苷酸为PS修饰的腺苷酸、PB修饰的腺苷酸、MP修饰的腺苷酸、LNA修饰的腺苷酸、PS修饰的腺苷酸与2’OMe修饰的腺苷酸的混合物、PS修饰的腺苷酸与PB修饰的腺苷酸、PS修饰的腺苷酸与MP修饰的腺苷酸、或PS修饰的腺苷酸与2’F修饰的腺苷酸的混合物，所述修饰胞苷酸为LNA修饰的胞苷酸。
  7. 如权利要求6所述的RNA分子，其特征在于，所述mRNA分子从5’到3’含有依次连接的5’非翻译区、Kozak序列、开放阅读框、3’非翻译区和所述poly(A)分子；
    优选地，所述开放阅读框编码感兴趣的蛋白质或多肽分子；优选地，所述蛋白质或多肽分子选自病原体抗原、肿瘤抗原、细胞因子、激素、抗体、嵌合抗原受体、酶和结构蛋白；
    优选地，所述病原体选自病毒、细菌、真菌、螺旋体和寄生虫；
    优选地，所述肿瘤抗原为肿瘤相关性抗原和肿瘤特异性抗原。
  8. 一种药物组合物，其特征在于，所述药物组合物含有权利要求5-7中任一项所述的RNA分子或采用权利要求1或2所述的方法制备得到的RNA分 子或采用权利要求3或4所述的方法制备得到的mRNA分子和药学上可接受的载体；
    优选地，所述药物组合物是疫苗，优选为预防性疫苗或治疗性疫苗，其中，预防性疫苗中所述RNA分子编码抗原多肽，治疗性疫苗中所述RNA分子编码治疗性多肽。
  9. 选自以下的应用：
    (1)修饰的ATP、修饰的CTP、修饰ATP和/或修饰的CTP与未修饰的ATP的混合物、或含修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸的poly(A)分子在制备胞内翻译效率和/或稳定性提高的已有poly(A)尾的RNA分子中的应用，或修饰的腺苷酸和/或修饰的胞苷酸或由修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子在提高已有poly(A)尾的RNA分子的胞内翻译效率和/或稳定性中的应用；优选地，所述修饰选自硫代磷酸修饰、硼代磷酸修饰、甲基代磷酸修饰、锁核酸修饰、2’甲氧基修饰和2’氟代修饰；
    (2)权利要求5-7中任一项所述的RNA分子或采用权利要求1或2所述的方法制备得到的RNA分子或采用权利要求3或4所述的方法制备得到的mRNA分子在制备核酸药物或核酸疫苗中的应用。
  10. 一种试剂盒，其特征在于，所述试剂盒含有修饰ATP和/或修饰CTP与未修饰ATP的混合物，或含有由修饰腺苷酸和/或修饰的胞苷酸和任选的未修饰腺苷酸组成的poly(A)分子，以及实施连接反应或聚合反应所需的试剂；
    优选地，所述修饰ATP和/或修饰CTP与未修饰ATP的混合物中，修饰的ATP和/或修饰CTP与未修饰ATP的比例在1∶19到19∶1的范围内，如在1∶9到9∶1或2∶8到8∶2的范围内；所述由修饰腺苷酸和/或修饰的胞苷酸与任选的未修饰腺苷酸组成的poly(A)分子不含有未修饰腺苷酸，或含有时，修饰的腺苷酸和/或修饰的胞苷酸与未修饰腺苷酸的比例在1∶19到19∶1的范围内，如在1∶9到9∶1或2∶8到8∶2的范围内；
    优选地，所述修饰选自硫代磷酸修饰、硼代磷酸修饰、甲基代磷酸修饰、锁核酸修饰、2’甲氧基修饰和2’氟代修饰。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1892299A2 (en) * 1997-09-19 2008-02-27 Sequitur, Inc. Sense mRNA therapy
WO2013013820A1 (en) * 2011-07-25 2013-01-31 Universität Heidelberg Functionalization of rna oligonucleotides
CN112673106A (zh) * 2017-12-21 2021-04-16 贝瑟克里科有限公司 点击修饰的mRNA
CN113195720A (zh) * 2018-10-12 2021-07-30 哈佛大学的校长及成员们 酶促rna合成

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1892299A2 (en) * 1997-09-19 2008-02-27 Sequitur, Inc. Sense mRNA therapy
WO2013013820A1 (en) * 2011-07-25 2013-01-31 Universität Heidelberg Functionalization of rna oligonucleotides
CN112673106A (zh) * 2017-12-21 2021-04-16 贝瑟克里科有限公司 点击修饰的mRNA
CN113195720A (zh) * 2018-10-12 2021-07-30 哈佛大学的校长及成员们 酶促rna合成

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANHÄUSER LEA, HÜWEL SABINE, ZOBEL THOMAS, RENTMEISTER ANDREA: "Multiple covalent fluorescence labeling of eukaryotic mRNA at the poly(A) tail enhances translation and can be performed in living cells", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 47, no. 7, 23 April 2019 (2019-04-23), GB , pages e42 - e42, XP093042985, ISSN: 0305-1048, DOI: 10.1093/nar/gkz084 *
STRZELECKA, D. ET AL.: "Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression", RNA, vol. 26, 20 August 2020 (2020-08-20), XP055929209, DOI: 10.1261/rna *

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