CN118021956A - MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof - Google Patents
MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof Download PDFInfo
- Publication number
- CN118021956A CN118021956A CN202211410072.4A CN202211410072A CN118021956A CN 118021956 A CN118021956 A CN 118021956A CN 202211410072 A CN202211410072 A CN 202211410072A CN 118021956 A CN118021956 A CN 118021956A
- Authority
- CN
- China
- Prior art keywords
- mrna
- monoclonal antibody
- seq
- sequence
- light chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 206010064097 avian influenza Diseases 0.000 title abstract description 10
- 208000002979 Influenza in Birds Diseases 0.000 title abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 25
- 108700026244 Open Reading Frames Proteins 0.000 claims abstract description 11
- 230000008488 polyadenylation Effects 0.000 claims abstract description 9
- 108020005345 3' Untranslated Regions Proteins 0.000 claims abstract description 6
- 108020003589 5' Untranslated Regions Proteins 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 239000013612 plasmid Substances 0.000 claims description 20
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 15
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 13
- 150000002632 lipids Chemical class 0.000 claims description 11
- 239000002105 nanoparticle Substances 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 9
- 150000007523 nucleic acids Chemical group 0.000 claims description 8
- 238000012163 sequencing technique Methods 0.000 claims description 7
- 230000009385 viral infection Effects 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 239000013600 plasmid vector Substances 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 108020004705 Codon Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 108091034057 RNA (poly(A)) Proteins 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 238000011321 prophylaxis Methods 0.000 claims description 2
- VZQHRKZCAZCACO-PYJNHQTQSA-N (2s)-2-[[(2s)-2-[2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]propanoyl]amino]prop-2-enoylamino]-3-methylbutanoyl]amino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)C(=C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCNC(N)=N VZQHRKZCAZCACO-PYJNHQTQSA-N 0.000 claims 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 claims 1
- 108091036066 Three prime untranslated region Proteins 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 2
- 230000004044 response Effects 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 10
- 230000003472 neutralizing effect Effects 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- -1 cationic lipid Chemical class 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 241000712461 unidentified influenza virus Species 0.000 description 7
- 241000700605 Viruses Species 0.000 description 6
- 108700021021 mRNA Vaccine Proteins 0.000 description 6
- 229940126582 mRNA vaccine Drugs 0.000 description 6
- 239000013598 vector Substances 0.000 description 5
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000005538 encapsulation Methods 0.000 description 4
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- 229930182558 Sterol Natural products 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000012827 research and development Methods 0.000 description 3
- 150000003432 sterols Chemical class 0.000 description 3
- 235000003702 sterols Nutrition 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- WTBFLCSPLLEDEM-JIDRGYQWSA-N 1,2-dioleoyl-sn-glycero-3-phospho-L-serine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC WTBFLCSPLLEDEM-JIDRGYQWSA-N 0.000 description 2
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 2
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 2
- UBCHPRBFMUDMNC-UHFFFAOYSA-N 1-(1-adamantyl)ethanamine Chemical compound C1C(C2)CC3CC2CC1(C(N)C)C3 UBCHPRBFMUDMNC-UHFFFAOYSA-N 0.000 description 2
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 description 2
- OGHAROSJZRTIOK-KQYNXXCUSA-O 7-methylguanosine Chemical compound C1=2N=C(N)NC(=O)C=2[N+](C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-O 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 2
- 229960003805 amantadine Drugs 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 2
- 210000001163 endosome Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical group O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 229960000402 palivizumab Drugs 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000003161 ribonuclease inhibitor Substances 0.000 description 2
- 229960000888 rimantadine Drugs 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- KYEKLQMDNZPEFU-KVTDHHQDSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazine-2,4-dione Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)N=C1 KYEKLQMDNZPEFU-KVTDHHQDSA-N 0.000 description 1
- MUSPKJVFRAYWAR-XVFCMESISA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)thiolan-2-yl]pyrimidine-2,4-dione Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)S[C@H]1N1C(=O)NC(=O)C=C1 MUSPKJVFRAYWAR-XVFCMESISA-N 0.000 description 1
- JCNGYIGHEUKAHK-DWJKKKFUSA-N 2-Thio-1-methyl-1-deazapseudouridine Chemical compound CC1C=C(C(=O)NC1=S)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O JCNGYIGHEUKAHK-DWJKKKFUSA-N 0.000 description 1
- CWXIOHYALLRNSZ-JWMKEVCDSA-N 2-Thiodihydropseudouridine Chemical compound C1C(C(=O)NC(=S)N1)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O CWXIOHYALLRNSZ-JWMKEVCDSA-N 0.000 description 1
- JUMHLCXWYQVTLL-KVTDHHQDSA-N 2-thio-5-aza-uridine Chemical compound [C@@H]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C(=S)NC(=O)N=C1 JUMHLCXWYQVTLL-KVTDHHQDSA-N 0.000 description 1
- VRVXMIJPUBNPGH-XVFCMESISA-N 2-thio-dihydrouridine Chemical compound OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)N1CCC(=O)NC1=S VRVXMIJPUBNPGH-XVFCMESISA-N 0.000 description 1
- GJTBSTBJLVYKAU-XVFCMESISA-N 2-thiouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=S)NC(=O)C=C1 GJTBSTBJLVYKAU-XVFCMESISA-N 0.000 description 1
- HOCJTJWYMOSXMU-XUTVFYLZSA-N 4-Methoxypseudouridine Chemical compound COC1=C(C=NC(=O)N1)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O HOCJTJWYMOSXMU-XUTVFYLZSA-N 0.000 description 1
- VTGBLFNEDHVUQA-XUTVFYLZSA-N 4-Thio-1-methyl-pseudouridine Chemical compound S=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 VTGBLFNEDHVUQA-XUTVFYLZSA-N 0.000 description 1
- AMMRPAYSYYGRKP-BGZDPUMWSA-N 5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1-ethylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)N(CC)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 AMMRPAYSYYGRKP-BGZDPUMWSA-N 0.000 description 1
- DDHOXEOVAJVODV-GBNDHIKLSA-N 5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=S)NC1=O DDHOXEOVAJVODV-GBNDHIKLSA-N 0.000 description 1
- BNAWMJKJLNJZFU-GBNDHIKLSA-N 5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4-sulfanylidene-1h-pyrimidin-2-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=S BNAWMJKJLNJZFU-GBNDHIKLSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001502567 Chikungunya virus Species 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- YKWUPFSEFXSGRT-JWMKEVCDSA-N Dihydropseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1C(=O)NC(=O)NC1 YKWUPFSEFXSGRT-JWMKEVCDSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 241000711920 Human orthopneumovirus Species 0.000 description 1
- 102000009617 Inorganic Pyrophosphatase Human genes 0.000 description 1
- 108010009595 Inorganic Pyrophosphatase Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- 108700040121 Protein Methyltransferases Proteins 0.000 description 1
- 229930185560 Pseudouridine Natural products 0.000 description 1
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 description 1
- 229940022005 RNA vaccine Drugs 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 description 1
- DGNMJYUPWDTKJB-ZDSKVHJSSA-N bis[(z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate Chemical compound CCCCCC\C=C/COC(=O)CCCCCCCC(OC(=O)CCCN(C)C)CCCCCCCC(=O)OC\C=C/CCCCCC DGNMJYUPWDTKJB-ZDSKVHJSSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002625 monoclonal antibody therapy Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- DWRXFEITVBNRMK-JXOAFFINSA-N ribothymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 DWRXFEITVBNRMK-JXOAFFINSA-N 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 229940126622 therapeutic monoclonal antibody Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Pulmonology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Optics & Photonics (AREA)
- Nanotechnology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to mRNA for encoding an anti-avian influenza H7N9 virus antibody, a preparation method and application thereof. Specifically, an isolated mRNA composition is provided that includes an mRNA comprising a monoclonal antibody heavy chain encoding H7N9 and an mRNA comprising a monoclonal antibody encoding H7N9, the mrnas further comprising: a 5' cap structure; a 5' UTR sequence; 3' UTR sequence and 4) polyadenylation sequence, the mass ratio of mRNA comprising the open reading frame encoding the H7N9 monoclonal antibody heavy chain to mRNA comprising the open reading frame encoding the H7N9 monoclonal antibody light chain in the composition is 1:1. The mRNA composition of the invention can be directly expressed as a monoclonal antibody in a subject, has quick response and can be directly used for treating H7N9 infection. And the effect is better than that of directly injecting monoclonal antibody.
Description
Technical Field
The invention belongs to the fields of immunology and molecular biology, and particularly relates to mRNA for encoding an anti-avian influenza H7N9 virus antibody, a preparation method and application thereof.
Background
The H7N9 virus is an avian influenza virus, has drug resistance to traditional antiviral drugs amantadine (amantadine) and rimantadine (rimantadine), and has no effective treatment means at present. In 2013, the H7N9 bird flu outbreak deprives lives of 40% of infected patients, and cities such as Shanghai shut down the live bird market, kill at least 17 ten thousand birds, and continue to prevail for years. The H7N9 virus needs to rely on specific molecules expressed by the virus itself to bind to receptors on human cells when invading the cells to infect the cells and further expand. The humanized antibody for neutralizing the virus is a certain specific antibody generated by human B lymphocytes, and can be combined with antigens on the surface of the virus, so that the virus is prevented from adhering to target cell receptors, the virus is prevented from invading cells, and the H7N9 influenza can be effectively prevented and treated. However, the high development and production costs of antibody drugs and the complex technical requirements of the neck limit their worldwide use. Therapeutic monoclonal antibodies are typically produced by cell lines, such as chinese hamster ovary cells, and then the antibodies are purified from the cell culture supernatant in large quantities and pharmaceutical formulations are developed. Production of antibodies presents a number of challenges, including misfolding or incorrect post-translational modifications that can lead to adverse events. The purification of each monoclonal antibody is specific and requires the development of a new method for each antibody, and thus the production cost is high.
In contrast, mRNA production and purification is simple, fast and cost effective because it does not require complex and expensive laboratory infrastructure, and the same method can be used for all mRNA. The sudden burst of new crown epidemic makes mRNA technology free and gets unprecedented attention. Nucleic acid-encoded monoclonal antibodies, particularly mRNA-based monoclonal antibodies, offer great promise for improved therapeutic efficacy and wide-ranging applications. The therapeutic mRNA vaccine has short research and development period, high safety and effectiveness, simple production process and high productivity, and is the most advanced biomedical technology at present. mRNA vaccines encoding neutralizing antibodies are an important trend in the development of drugs for the treatment of infectious diseases: mRNA encoding a broad spectrum neutralizing HIV antibody VRC01 successfully produced VRC01 in vivo after injection into mice and protected human mice from HIV-1 virus infection; aiming at human RSV, tiwari develops the prior drug palivizumab into mRNA encoding membrane anchoring neutralizing antibody, which has higher efficiency than palivizumab and significantly inhibits RSV after 7 days of transfection; mRNA encoding the chikungunya virus neutralizing antibody was efficiently expressed in mice and protected from arthritis and musculoskeletal tissue infection 2 days after inoculation, and viremia was reduced to undetectable levels. Anti-avian influenza virus monoclonal antibodies can prevent and treat avian influenza virus infection. However, the antibody is subject to purification and post-translational modification during cell production, and is expensive to develop and produce, so that the use of mRNA technology encoding anti-avian influenza antibodies for more efficient, safe, and low-cost monoclonal antibody therapy is a new approach to preventing and treating avian influenza.
Antibody drugs are special-effect drugs for treating serious infectious diseases, but their high research, development and production costs and the complex technical requirements of the neck limit their worldwide application. The mRNA drug research and development period is short, the safety and the effectiveness are high, the production process is simple, the productivity is high, and the method is the most advanced biotechnology at present. Currently, no mRNA vaccine against H7N9 virus infection is seen, and there is a few direct uses of monoclonal antibody mRNA expressing neutralizing virus as a therapeutic mRNA vaccine for inhibiting H7N9 infection.
Disclosure of Invention
To solve the above problems, the inventors of the present invention utilized the sequence of a monoclonal antibody (bulletin number: CN_110746503_B) against H7N9 avian influenza virus developed in the previous study. The invention transcribes this antibody gene into mRNA and wraps it in LNP to form an LNP-RNA vaccine, which is capable of treating the H7N9 virus after vaccination of a subject.
The first aspect of the invention provides an isolated mRNA composition comprising mRNA comprising a heavy chain of a monoclonal antibody encoding H7N9 and mRNA comprising a light chain of a monoclonal antibody encoding H7N9,
The amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the H7N9 monoclonal antibody are shown below, respectively:
heavy chain CDR1 region: GYIFTSYD SEQ ID No.1;
heavy chain CDR2 region: MNPDSGDT SEQ ID No.2;
Heavy chain CDR3 region: ATGNADCSGGSCYNWFDP SEQ ID No.3;
light chain CDR1 region: RLRSYY SEQ ID No.4;
light chain CDR2 region: GKN;
Light chain CDR3 region: NSRDTSGYHLV SEQ ID No.5;
The mRNAs also respectively comprise:
1) A 5' cap structure;
2) A 5' UTR sequence;
3) Sequence encoding a Signal peptide
4) Stop codon and enzyme cutting site sequence
5) 3' UTR sequences; and
6) A poly (A) sequence of a poly (A),
Wherein, the mRNA containing the heavy chain of the monoclonal antibody encoding H7N9 or the mRNA containing the light chain of the monoclonal antibody encoding H7N9 respectively comprises the following elements in sequence along the 5 '. Fwdarw.3' direction: a 5' cap structure, a 5' UTR sequence, a sequence encoding a signal peptide, mRNA corresponding to the open reading frame of the heavy or light chain of a monoclonal antibody of H7N9, a stop codon and cleavage site sequence, a 3' UTR sequence and a polyadenylation sequence.
Further, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 6;
QVQLVESGAEVKKPGASVKVSCKASGYIFTSYDINWVRQATGQGLEWMGWMNPDSGDTGFAQKFQGRVTMTRNTSITTAYMELSSLTSEDTAVYYCATGNADCSGGSCYNWFDPWGQGTLVTVSS SEQ ID NO.6; And/or
The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 7:
SSELTQDPAVSVALGQTVRITCQGDRLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDTSGYHLVFGGGTKLTVLV SEQ ID NO.7.
further, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 8;
QVQLVESGAEVKKPGASVKVSCKASGYIFTSYDINWVRQATGQGLEWMGWMNPDSGDTGFAQKFQGRVTMTRNTSITTAYMELSSLTSEDTAVYYCATGNADCSGGSCYNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.8; And/or
The light chain amino acid sequence of the antibody is shown as SEQ ID NO. 9:
SSELTQDPAVSVALGQTVRITCQGDRLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDTSGYHLVFGGGTKLTVLVTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.9.
further, the RNA sequence corresponding to the open reading frame of the heavy chain of the monoclonal antibody of H7N9 is the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO. 10:
caagtgcagctggtggagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacatattcaccagttatgatatcaactgggtgcgacaggccactggccaagggcttgagtggatgggatggatgaaccctgacagtggtgacacaggctttgcacagaagttccagggcagagtcaccatgaccaggaacacctccataaccacagcctacatggagctgagcagcctgacttctgaggacacggccgtgtattactgtgcgacaggaaatgcggattgtagtggtggtagctgctacaattggttcgacccctggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggccgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga SEQ ID NO.10
The RNA sequence corresponding to the open reading frame of the H7N9 monoclonal antibody light chain is the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO. 11:
tcgtctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagactcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacaccagtggttaccatctggtgttcggcggagggaccaagctgaccgtcctagtaaccgtggccgccccctccgtgttcatcttccccccctccgacgagcagctgaagtccggcaccgcctccgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggagtccgtgaccgagcaggactccaaggactccacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaggttacccaccagggcctgtcctcccccgtgaccaagtccttcaaccggggcgagtgctag
SEQ ID NO.11
Further, the 5 'cap structure is selected from at least one of m7GpppG, m27,3' -ogppg, m7Gppp (5 ') N1 or m7Gppp (m 2' -O) N1.
Further, the 5' UTR sequence is selected from the group consisting of RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID NO. 12.
ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC SEQ ID NO.12。
In other embodiments of the invention, the 3' UTR sequence is selected from the group consisting of the RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID NO. 13.
CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC SEQ ID NO.13
Further, the polyadenylation sequence comprises a sequence of 25-400 adenylates.
Further, the polyadenylation sequence comprises a sequence of 50-400 adenylates.
Further, the polyadenylation sequence comprises a sequence of 50-300 adenylates.
Further, the polyadenylation sequence comprises a sequence of 50-250 adenylates.
Further, the polyadenylation sequence comprises a sequence of 80-120 adenylates.
Further, the sequence encoding the signal peptide is selected from the group consisting of RNA sequences corresponding to the sequence shown in SEQ ID NO. 16.
Further, the cleavage site sequence is selected from the group consisting of the RNA sequences corresponding to CTCGAG.
In a second aspect the invention provides a pharmaceutical composition comprising the mRNA composition of the first aspect, and optionally a delivery vehicle.
Further, the delivery vehicle is a nanoparticle.
Further, the delivery vehicle is a lipid nanoparticle.
In other embodiments of the present invention, the lipid nanoparticle comprises a cationic lipid and at least one selected from the group consisting of a non-cationic lipid, a sterol, a PEG-modified lipid.
In some embodiments of the invention, the lipid nanoparticle is a cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid.
In other embodiments of the invention, the cationic lipid is an ionizable cationic lipid selected from one or more of the following: 2, 2-Di-lino-4-dimethylaminoethyl- [1,3] -dioxolane, di-lino-methyl-4-dimethylaminobutyrate and 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioic acid di ((Z) -non-2-en-1-yl) ester, preferably Di-lino-methyl-4-dimethylaminobutyrate. In some embodiments of the invention, the non-cationic lipid is a neutral lipid selected from at least one of distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl lecithin (DOPC), and dioleoyl phosphatidylserine (DOPS).
In other embodiments of the invention, the sterol is cholesterol.
In some embodiments of the invention, the PEG-modified lipid is selected from at least one of PEG-DMG, PEG-DSG, and PEG-DMPE.
In other embodiments of the invention, the PEG-modified lipid has a PEG length of 0.5-200KDa.
In some embodiments of the invention, the pharmaceutical composition optionally contains an adjuvant.
The third aspect of the invention provides a kit comprising an mRNA composition according to the first aspect of the invention and/or a pharmaceutical composition according to the second aspect of the invention.
According to a fourth aspect of the invention, there is provided an mRNA composition according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, and the use of a kit according to the third aspect of the invention in the manufacture of a medicament for the prophylaxis and/or treatment of H7N9 virus infection diseases.
A fifth aspect of the present invention provides a method for producing the mRNA composition according to the first aspect of the present invention, comprising the steps of:
S1) respectively inserting heavy chain genes and light chain genes of the H7N9 monoclonal antibody into plasmid vectors to obtain plasmid vectors containing the heavy chain genes or the light chain genes of the H7N9 monoclonal antibody;
S2) respectively transferring the plasmid vectors containing the H7N9 monoclonal antibody heavy chain and light chain genes obtained in the step 1) into host bacteria for culture and sequencing;
s3) carrying out amplification culture on the monoclonal with correct sequencing in the step 2), and extracting plasmids;
s4) obtaining a linearization plasmid by enzyme digestion of the plasmid;
S5) transcribing the linearized plasmid into RNA;
S6) capping the RNA obtained in the step S5) to obtain mRNA,
S7) mRNA comprising the open reading frames of the H7N9 monoclonal antibody heavy and light chain genes were mixed at 1:1 to obtain mRNA compositions.
The invention has the beneficial effects that:
The H7N9 mRNA vaccine has a short research and development period, and is particularly suitable for developing new sudden infectious disease vaccines including H7N 9; the safety is high. The mRNA composition of the invention overcomes the problems possibly occurring in the prior art when antigen mRNA vaccines are used for treating virus infection diseases, such as different immune response degrees of different subjects after inoculation, not all subjects can produce corresponding antibodies, even if the antibodies are produced, the antibody production time is longer, and the problem that the yield is insufficient to inhibit virus infection can also occur. The mRNA composition can be directly expressed as a monoclonal antibody in a subject, has quick response, can be directly used for treating H7N9 infection, and has better effect than the direct injection of the monoclonal antibody.
Drawings
FIG. 1 is a photograph of plasmid gel electrophoresis.
FIG. 2 is a photograph of transcribed mRNA gel electrophoresis.
FIG. 3 is an ELISA to verify antibody expression of LNP-mRNA in cells.
Detailed Description
The following detailed description of the present invention will be made in detail to make the above objects, features and advantages of the present invention more apparent, but should not be construed to limit the scope of the present invention.
In some embodiments of the invention, the mRNA is not chemically modified or is chemically modified, the chemical modification being a replacement of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in the mRNA, replacement of uracil with pseudouridine, N1-methyl-pseudouridine, N1-ethyl-pseudouridine, 2-thiouridine, 4' -thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine or 5-methoxy-1-methyl-uridine, and N-methyl-1-methyl-pseudouridine or further preferably N1-methyl-pseudouridine;
In some embodiments of the invention, the 5 'cap structure is selected from at least one of m7GpppG, m27,3' -OGpppG, m7Gppp (5 ') N1 or m7Gppp (m 2' -O) N1.
According to the demands of different mRNAs, different 5 'cap structures can be flexibly added at the 5' end of the mRNAs.
"M7G" represents a 7-methylguanosine capped nucleoside, "ppp" represents a triphosphate bond between the 5 'carbon of the capped nucleoside and the first nucleotide of the primary RNA transcript, N1 is the most 5' nucleotide, "G" represents a guanosine, "m7" represents a methyl group at the 7-position of guanine, and "m2'-O" represents a methyl group at the 2' -O position of the nucleotide.
The nanoparticle provided by the invention can be used for efficiently delivering mRNA and has the following characteristics and advantages: for example, in the encapsulation of mRNA, acidic pH conditions cause the ionizable cationic lipid to carry a positive charge, compressing the negatively charged mRNA molecule, and thus achieving higher encapsulation efficiency; under the physiological pH condition, the ionizable lipid nanoparticle has neutrality, does not act with negatively charged cell membranes, and has high biocompatibility; after the ionizable lipid nanoparticle enters cells through endocytosis to form endosomes, the acidic condition in the endosomes enables the nanoparticle to be positively charged again and electrostatically interact with the endosomal membrane with negative charges, so that mRNA release is facilitated.
Example 1 preparation of mRNA
(1) The neutralizing avian influenza virus antibody gene was inserted into pUC57-kana vector: the H7N9 antibody heavy chain gene, the H7N9 antibody light chain gene and the pUC57 vector (stored in this laboratory) were double digested with restriction enzymes EcoR1 and Hind3 (purchased from Thermo), respectively, and the antibody heavy chain gene and the antibody light chain gene were ligated by using T4 ligase (Thermo), respectively; obtaining a vector connected with a heavy chain gene of the antibody of the neutralizing avian influenza virus and a vector connected with the light chain gene of the antibody of the neutralizing avian influenza virus.
The H7N9 antibody heavy chain gene is shown as SEQ ID NO.10
caagtgcagctggtggagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacatattcaccagttatgatatcaactgggtgcgacaggccactggccaagggcttgagtggatgggatggatgaaccctgacagtggtgacacaggctttgcacagaagttccagggcagagtcaccatgaccaggaacacctccataaccacagcctacatggagctgagcagcctgacttctgaggacacggccgtgtattactgtgcgacaggaaatgcggattgtagtggtggtagctgctacaattggttcgacccctggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggccgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga SEQ ID NO.10.
The heavy chain gene sequence of the H7N9 antibody is encoded before the heavy chain gene sequence, and the heavy chain gene sequence also comprises a signal peptide sequence SEQ ID NO.16; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC SEQ ID No.16. The signal peptide can guide the newly synthesized protein to transfer to the secretion passage after being expressed, and the signal peptide used in the invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
Other gene sequences capable of encoding H7N9 antibody heavy chain SEQ ID NO.8 may also be used. The heavy chain sequence of the H7N9 antibody is shown in SEQ ID NO. 8:
QVQLVESGAEVKKPGASVKVSCKASGYIFTSYDINWVRQATGQGLEWMGWMNPDSGDTGFAQKFQGRVTMTRNTSITTAYMELSSLTSEDTAVYYCATGNADCSGGSCYNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.8.
the H7N9 antibody light chain gene is shown in SEQ ID NO. 11:
tcgtctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagactcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacaccagtggttaccatctggtgttcggcggagggaccaagctgaccgtcctagtaaccgtggccgccccctccgtgttcatcttccccccctccgacgagcagctgaagtccggcaccgcctccgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggagtccgtgaccgagcaggactccaaggactccacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaggttacccaccagggcctgtcctcccccgtgaccaagtccttcaaccggggcgagtgctagSEQ ID NO.11.
The sequence of the light chain gene of the H7N9 antibody is encoded and also comprises a signal peptide sequence SEQ ID NO.16; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC SEQ ID No.16. The signal peptide can guide the newly synthesized protein to transfer to the secretion passage after being expressed, and the signal peptide used in the invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
Other gene sequences capable of encoding the H7N9 antibody light chain SEQ ID NO.9 may also be used. The H7N9 antibody light chain sequence is shown in SEQ ID NO. 9:
SSELTQDPAVSVALGQTVRITCQGDRLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDTSGYHLVFGGGTKLTVLVTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.9.
(2) Vector transformation XL1-blue strain: taking a tube of competent cells, adding 1 mu L (50 ng) of the plasmid containing the light chain gene or the plasmid containing the heavy chain gene obtained in the step (1), and uniformly mixing; ice bath for 5min, heat shock for 90 s, and ice placement for 5 min; 500. Mu.L of 2YT liquid medium was added at 37℃for 45 minutes at 200 rpm; plating by sucking 50 mu L of bacterial liquid and culturing overnight; selecting the monoclonal and sending to Jin Weizhi company for sequencing; the sequencing result is shown as SEQ ID NO.14 and SEQ ID NO.15 :ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCcaagtgcagctggtggagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacatattcaccagttatgatatcaactgggtgcgacaggccactggccaagggcttgagtggatgggatggatgaaccctgacagtggtgacacaggctttgcacagaagttccagggcagagtcaccatgaccaggaacacctccataaccacagcctacatggagctgagcagcctgacttctgaggacacggccgtgtattactgtgcgacaggaaatgcggattgtagtggtggtagctgctacaattggttcgacccctggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggccgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAASEQ ID NO.14;
ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCtcgtctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagactcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacaccagtggttaccatctggtgttcggcggagggaccaagctgaccgtcctagtaaccgtggccgccccctccgtgttcatcttccccccctccgacgagcagctgaagtccggcaccgcctccgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggagtccgtgaccgagcaggactccaaggactccacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaggttacccaccagggcctgtcctcccccgtgaccaagtccttcaaccggggcgagtgctagTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO.15;
(3) Monoclonal expansion culture, plasmid extraction: the correctly sequenced monoclonal was cultured in 400ml of 2YT medium at 37℃overnight. Extracting plasmid by using CWBIO company plasmid extraction kit to obtain 5.7mg plasmid, and sequencing correctly;
(4) Plasmid linearization: plasmid was digested with Bsa I, and plasmid (2887. Mu.L), enzyme (333. Mu.L), buffer (1665. Mu.L) and DEPC water (11765. Mu.L) were added in this order to the centrifuge tube, followed by a water bath at 37℃overnight. Liquid is exchanged and recovered by a 100K ultrafiltration tube, and the concentration of the linearized plasmid is 358.1 ng/. Mu.L;
(5) Transcription: into the centrifuge tube were added 412. Mu.L of plasmid, 320. Mu.L of buffer, 64. Mu.L of ATP, 64. Mu.L of GTP, 64. Mu.L of CTP, 64. Mu.L of pUTP, 160. Mu.L of RNase inhibitor, 3.2. Mu.L of inorganic pyrophosphatase, 160. Mu. L T7 RNA polymerase, 1888.8. Mu.L of DEPC water. The reaction was carried out at 37℃for 3 hours. Adding 160 mu L DNase I enzyme for digestion for 15 minutes; adding 4800 mu L of LiCl precipitation solution and 4800 mu L of DEPC water, uniformly mixing, and standing in a refrigerator at-20 ℃ for 30 minutes; centrifuging at 12000rpm for 15 minutes, cleaning with 70% ethanol for 2 times, and removing residual ethanol in the centrifuge tube as much as possible; 1500 μl of water was added to dissolve RNA. 5101 ng/. Mu.L RNA was finally obtained, together with 7.22mg;
(6) Capping m7Gppp (5') N1: 596. Mu.L of RNA, 3424. Mu.L of water, and denaturing at 65℃for 5 minutes are added to the centrifuge tube; 600. Mu.L buffer, 600. Mu.L GTP, 150. Mu.L SAM, 150. Mu.L RNase inhibitor, 240. Mu.L capping enzyme were added; 240. Mu.L of methyltransferase, and reacted at 37℃for 1 hour; adding 9mL of LiCl precipitation solution and 9mLDEPC of water, uniformly mixing, and standing in a refrigerator at-20 ℃ for 30min; centrifuging at 12000rpm for 15 minutes, cleaning with 70% ethanol for 2 times, and removing residual ethanol in the centrifuge tube as much as possible; the cover is opened and placed in an ultra clean bench for airing, and 3000 mu L of water is added for dissolving RNA. The Nanodrop detection concentration was 721ng/mL, yield 2.095mg.
(7) Preparation of mRNA mixture: mRNA containing the heavy chain and light chain gene open reading frames of the H7N9 monoclonal antibody were mixed at a mass ratio of 1:1, respectively, to obtain mRNA compositions.
EXAMPLE 2 preparation of LNP Encapsulated mRNA
The experiment of LNP encapsulation of mRNA was completed by offshore technology Co., ltd, LNP-mRNA was milky white in appearance with an encapsulation rate >98%, dispersion coefficient of 0.11, and nanoparticle size of 92.84nm.
LNP-mRNA was transfected into 293T cells using LIPO3000 (Invitrogen), after 48 hours, the supernatant and cell lysate were collected, 100. Mu.L/well of the coated ELISA plates were diluted 20 times with PBS, incubated 2 hours at 37℃for 2 times, PBS washed 2 times, 5% nonfat dry milk blocked for 1 hour, PBS washed 2 times, 100. Mu.L of anti-his tag antibody was added to each well, incubated 1 hour at 37℃for 3 times, 100. Mu.L of chromogenic solution was added to each well for 10 minutes, and finally 100. Mu.L of stop solution was added, and OD450 data were recorded on a spectrophotometer.
The results are shown in FIG. 3: cells transfected with LNP-mRNA expressed the antibody of interest in the supernatant.
Example 3
Sodium pentobarbital solution with final concentration of 1% is prepared with physiological saline, filtered by 0.22 μm filter, and stored at low temperature of 4deg.C. The mice were weighed and the anesthetized mice were intraperitoneally dosed at 150 μl/20g and were nasally infected with 20 μl of 10×LD 50-containing H7N9 virus. After 12 hours, mice were randomly divided into PBS group, LNP-mRNA experimental group and antibody experimental group, 8 mice each, and tail vein was inoculated with PBS, LNP-mRNA (1.4 mg/kg) and antibody (20 mg/kg), respectively. The day of nasal drip was day 0 of the mouse experiment. From the day of infection, the number of mice survived was recorded until 2 weeks after infection
The results show that: animals in the PBS group all died, mice in the LNP-mRNA group survived 87.5% and mice in the antibody-treated group survived 75%. LNP-mRNA encoding the antibody at lower doses was able to protect mice more significantly from lethal challenge with H7N9 virus than in the antibody-treated group. LNP-mRNA encoding anti-avian influenza antibodies is capable of expressing antibodies directly in vivo and significantly protecting mice from lethal challenge with H7N9 virus.
Claims (10)
1. An isolated mRNA composition comprising mRNA comprising a heavy chain of a monoclonal antibody encoding H7N9 and mRNA comprising a light chain of a monoclonal antibody encoding H7N9,
The heavy and light chains of the H7N9 monoclonal antibody have the CDR1, CDR2 and CDR3 regions as shown below:
heavy chain CDR1 region: GYIFTSYD SEQ ID No.1;
heavy chain CDR2 region: MNPDSGDT SEQ ID No.2;
Heavy chain CDR3 region: ATGNADCSGGSCYNWFDP SEQ ID No.3;
light chain CDR1 region: RLRSYY SEQ ID No.4;
Light chain CDR2 region: GKN;
Light chain CDR3 region: NSRDTSGYHLV SEQ ID No.5;
The mRNA comprising the heavy chain of the monoclonal antibody encoding H7N9 or the mRNA comprising the light chain of the monoclonal antibody encoding H7N9, respectively, further comprises:
1) A 5' cap structure;
2) A 5' UTR sequence;
3) Sequence encoding a Signal peptide
4) Stop codon and enzyme cutting site sequence
5) 3' UTR sequences; and
6) A poly (A) sequence of a poly (A),
Wherein, the mRNA containing the heavy chain of the monoclonal antibody encoding H7N9 or the mRNA containing the light chain of the monoclonal antibody encoding H7N9 sequentially comprises the following elements in the 5 '. Fwdarw.3' direction: a 5' cap structure, a 5' UTR sequence, a sequence encoding a signal peptide, an RNA sequence corresponding to the open reading frame of the heavy or light chain of a monoclonal antibody encoding H7N9, a stop codon and cleavage site sequence, a 3' UTR sequence and a polyadenylation sequence;
Preferably, the 5 'cap structure is selected from at least one of m7GpppG, m27,3' -ogppg, m7Gppp (5 ') N1 or m7Gppp (m 2' -O) N1;
Preferably, the polyadenylation sequence comprises a sequence of 25-400 adenylates;
Preferably, the mass ratio of the mRNA comprising the heavy chain of the monoclonal antibody encoding H7N9 to the mRNA comprising the light chain of the monoclonal antibody encoding H7N9 in the composition is 1:1.
2. The isolated mRNA composition of claim 1, wherein the heavy chain variable region amino acid sequence of the monoclonal antibody to H7N9 is set forth in SEQ ID No. 6; and/or
The amino acid sequence of the light chain variable region of the monoclonal antibody of H7N9 is shown in SEQ ID NO. 7:
Preferably, the heavy chain amino acid sequence of the H7N9 monoclonal antibody is shown as SEQ ID NO. 8; and/or
The light chain amino acid sequence of the H7N9 monoclonal antibody is shown as SEQ ID NO. 9.
3. The isolated mRNA composition of any one of claims 1-2, wherein the open reading frame nucleic acid sequence encoding the heavy chain of the monoclonal antibody of H7N9 is set forth in SEQ ID No. 10;
The open reading frame nucleic acid sequence of the monoclonal antibody light chain of the coding H7N9 is shown in SEQ ID NO. 11.
4. The isolated mRNA composition of any one of claims 1-2, wherein the 5' utr sequence is selected from the group consisting of RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID No. 12.
5. The isolated mRNA composition of any one of claims 1-3, wherein the 3' utr sequence is selected from the group consisting of RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID No. 13.
6. A pharmaceutical composition, characterized in that it comprises the mRNA composition of any one of claims 1 to 5, and optionally a delivery vehicle;
preferably, the delivery vehicle is a nanoparticle;
Preferably, the delivery vehicle is a lipid nanoparticle.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition optionally contains an adjuvant.
8. Kit, characterized in that it comprises an mRNA composition according to any one of claims 1 to 5 and/or a pharmaceutical composition according to claim 6 or 7.
9. Use of an mRNA composition according to any one of claims 1 to 5, an mRNA composition according to claim 6 or 7, a pharmaceutical composition according to claim 8, or a kit according to claim 8 for the preparation of a medicament for the prophylaxis and/or treatment of H7N9 virus infection diseases.
10. The method for preparing an mRNA composition according to any one of claims 1 to 5, wherein the method comprises the steps of:
S1) respectively inserting heavy chain genes and light chain genes of the H7N9 monoclonal antibody into plasmid vectors to obtain plasmid vectors containing the heavy chain genes or the light chain genes of the H7N9 monoclonal antibody;
S2) respectively transferring the plasmid vectors containing the H7N9 monoclonal antibody heavy chain and light chain genes obtained in the step 1) into host bacteria for culture and sequencing;
s3) carrying out amplification culture on the monoclonal with correct sequencing in the step 2), and extracting plasmids;
s4) obtaining a linearization plasmid by enzyme digestion of the plasmid;
S5) transcribing the linearized plasmid into RNA;
S6) capping the RNA obtained in the step S5) to obtain mRNA,
S7) mRNA comprising the open reading frames of the H7N9 monoclonal antibody heavy and light chain genes were mixed at 1:1 to obtain mRNA compositions.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211410072.4A CN118021956A (en) | 2022-11-11 | 2022-11-11 | MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof |
PCT/CN2022/138182 WO2024098498A1 (en) | 2022-11-11 | 2022-12-09 | Mrna for encoding anti-avian influenza h7n9 virus antibody, preparation method therefor, and use thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211410072.4A CN118021956A (en) | 2022-11-11 | 2022-11-11 | MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN118021956A true CN118021956A (en) | 2024-05-14 |
Family
ID=90997336
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211410072.4A Pending CN118021956A (en) | 2022-11-11 | 2022-11-11 | MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN118021956A (en) |
WO (1) | WO2024098498A1 (en) |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018232355A1 (en) * | 2017-06-15 | 2018-12-20 | Modernatx, Inc. | Rna antibodies |
CN110746503B (en) * | 2018-07-24 | 2022-06-03 | 深圳先进技术研究院 | anti-H7N 9 fully human monoclonal antibody hIg311, preparation method and application thereof |
US20230272052A1 (en) * | 2020-07-31 | 2023-08-31 | CureVac SE | Nucleic acid encoded antibody mixtures |
IL302631A (en) * | 2020-11-06 | 2023-07-01 | Sanofi Sa | Lipid nanoparticles for delivering mrna vaccines |
EP4330404A1 (en) * | 2021-04-28 | 2024-03-06 | Genevant Sciences Gmbh | Mrna delivery constructs and methods of using the same |
-
2022
- 2022-11-11 CN CN202211410072.4A patent/CN118021956A/en active Pending
- 2022-12-09 WO PCT/CN2022/138182 patent/WO2024098498A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
WO2024098498A1 (en) | 2024-05-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220202934A1 (en) | Respiratory virus nucleic acid vaccines | |
US10709779B2 (en) | Nucleic acid vaccines | |
US11351242B1 (en) | HMPV/hPIV3 mRNA vaccine composition | |
CN113151312B (en) | Novel coronavirus SARS-CoV-2mRNA vaccine and its preparation method and application | |
TW202222821A (en) | Compositions and methods for the prevention and/or treatment of covid-19 | |
WO2023137550A1 (en) | Compositions and methods for the prevention and/or treatment of covid-19 | |
WO2022245888A1 (en) | Seasonal flu rna vaccines and methods of use | |
WO2023098679A1 (en) | Novel coronavirus mrna vaccine against mutant strains | |
CN118021956A (en) | MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof | |
CN118021957A (en) | MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof | |
CA3118329A1 (en) | Compositions and methods for the prevention and/or treatment of covid-19 | |
JP7504464B2 (en) | Nucleic Acid Vaccines | |
WO2024051266A1 (en) | Mrna for expressing varicella-zoster virus antigen protein and use thereof | |
TW202330923A (en) | Compositions and methods for the prevention and/or treatment of covid-19 | |
WO2023037320A1 (en) | Mucosal messenger rna vaccine | |
CN116655749A (en) | Rta truncated mRNA related vaccine of EBV and preparation method and application thereof | |
CA3107232A1 (en) | Compositions and methods for the prevention and/or treatment of covid-19 | |
CN117625651A (en) | Rabies virus mRNA vaccine and application thereof | |
WO2024073848A1 (en) | Compositions and methods for the prevention and/or treatment of covid-19 | |
TWI302166B (en) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |