WO2024143440A1 - アデノ随伴ウイルスの製造方法、およびベクターのセット - Google Patents

アデノ随伴ウイルスの製造方法、およびベクターのセット Download PDF

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Publication number
WO2024143440A1
WO2024143440A1 PCT/JP2023/046847 JP2023046847W WO2024143440A1 WO 2024143440 A1 WO2024143440 A1 WO 2024143440A1 JP 2023046847 W JP2023046847 W JP 2023046847W WO 2024143440 A1 WO2024143440 A1 WO 2024143440A1
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Prior art keywords
vector
gene
promoter
rep
cells
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Ceased
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PCT/JP2023/046847
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English (en)
French (fr)
Japanese (ja)
Inventor
太一 村口
勇介 森
裕輝 西川
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Fujifilm Corp
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Fujifilm Corp
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Priority to JP2024567906A priority Critical patent/JPWO2024143440A1/ja
Priority to EP23912218.7A priority patent/EP4644556A4/en
Publication of WO2024143440A1 publication Critical patent/WO2024143440A1/ja
Anticipated expiration legal-status Critical
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    • 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
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/50—Vectors for producing vectors
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • ⁇ 1> a gene transfer step of transferring a first vector containing a Rep gene and a Cap gene and a second vector containing a Rep gene and a Cap gene into a cell; and a cell culture step of culturing the gene-transfected cells,
  • the first vector and the second vector are different vectors, the Rep genes contained in the first vector and the second vector are full-length and of the same serotype;
  • the Cap gene contained in the first vector and the second vector is full-length and of the same serotype.
  • ⁇ 3> The method according to ⁇ 1> or ⁇ 2>, wherein the promoter of the first vector is a wild-type promoter of an adeno-associated virus.
  • the promoter of the first vector is a P5 promoter, a P19 promoter, or a P40 promoter.
  • the promoter of the second vector is a non-adeno-associated virus promoter.
  • ⁇ 6> The method according to ⁇ 1> or ⁇ 2>, wherein the promoter of the second vector is a CMV promoter, a PGK promoter, or an EF1 promoter.
  • ⁇ 10> The method according to ⁇ 1> or ⁇ 2>, wherein the amount of the second vector used is 5% or more based on the total amount of the first vector and the second vector used.
  • the first vector and the second vector are plasmid vectors.
  • ⁇ 12> The method according to ⁇ 1> or ⁇ 2>, wherein in the cell introduction step, a third vector containing a viral helper gene and a fourth vector containing a therapeutic or preventive gene are introduced into the cell.
  • ⁇ 13> The method according to ⁇ 1> or ⁇ 2>, wherein the cell is a HEK293 cell.
  • the present invention relates to a method for producing an adeno-associated virus, which includes a gene transfer step of transferring a first vector containing a Rep gene and a Cap gene and a second vector containing a Rep gene and a Cap gene into a cell, and a cell culture step of culturing the cells into which the genes have been transferred, wherein the first vector and the second vector are different vectors, the Rep genes contained in the first vector and the second vector are full-length and of the same serotype, and the Cap genes contained in the first vector and the second vector are full-length and of the same serotype.
  • the Rep gene contained in the first vector and the second vector, and the Cap gene contained in the first vector and the second vector are each full length.
  • Full length means the sequence from the start codon (the codon encoding Met) to the stop codon of the Rep gene.
  • the Rep genes contained in the first vector and the second vector, and the Cap genes contained in the first vector and the second vector, are of the same serotype.
  • the same serotype means that the AAV serotypes from which the Rep genes and the Cap genes are derived are the same.
  • the Rep gene and Cap gene which are genes necessary for the production of adeno-associated virus (AAV) are introduced into cells using two types of vectors (a first vector and a second vector).
  • AAV adeno-associated virus
  • the cells are not particularly limited, but are preferably animal cells, preferably mammalian cells or insect cells, and more preferably mammalian cells.
  • Mammalian cells include, but are not particularly limited to, human cells, mouse cells, rat cells, monkey cells, hamster cells, etc.
  • human cells can be used.
  • cells examples include mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2/0, YB2/0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK293 (human embryonic kidney cells), VERO, PER. C6, HeLa, EB1, EB2, EB3, oncolytic or hybridoma cell lines.
  • the cells are HEK293 cells or CHO cells, more preferably HEK293 cells.
  • HEK293 cells are cells into which the E1A and E1B genes derived from adenovirus are incorporated.
  • the cells may be either adherent cells or suspension cells, but suspension cells are preferred for high-density, large-scale culture in a reactor.
  • Thermo Fisher's suspension HEK293 cell line Viral Production Cells 2.0 (also referred to as VPCs 2.0) can be used.
  • the cell concentration at the time of gene introduction is preferably 1 x 10 cells/mL to 100 x 10 cells/mL, more preferably 1 x 10 cells/mL to 50 x 10 cells/mL, and even more preferably 2 x 10 cells/mL to 10 x 10 cells/mL.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2023/046847 2022-12-28 2023-12-27 アデノ随伴ウイルスの製造方法、およびベクターのセット Ceased WO2024143440A1 (ja)

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JP2024567906A JPWO2024143440A1 (https=) 2022-12-28 2023-12-27
EP23912218.7A EP4644556A4 (en) 2022-12-28 2023-12-27 PROCESS FOR PRODUCING ADENE-ASSOCIATED VIRUS, AND VECTOR SET

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JP2022-211032 2022-12-28

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120249230A (zh) * 2025-06-05 2025-07-04 鼐济医药科技(杭州)有限公司 一种用于同时生产多种重组腺相关病毒的方法

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JP2010534472A (ja) 2007-07-26 2010-11-11 アムステルダム モレキュラー セラピューティクス ビー．ブイ． 差次的コドンバイアスを有する反復コード配列を含むバキュロウイルスベクター
JP2011512156A (ja) * 2008-02-19 2011-04-21 アムステルダム モレキュラー セラピューティクス ビー．ブイ． 昆虫細胞におけるパルボウイルスｒｅｐ及びｃａｐタンパク質の発現の最適化
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WO2019057691A1 (en) 2017-09-19 2019-03-28 Cevec Pharmaceuticals Gmbh INDAVTIBLE AAV GEN GENES
JP2019513401A (ja) * 2016-04-15 2019-05-30 ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア 新規なａａｖ８変異カプシド及びそれを含有する組成物
JP2021510496A (ja) 2018-01-19 2021-04-30 オックスフォード ジェネティクス リミテッドＯｘｆｏｒｄ Ｇｅｎｅｔｉｃｓ Ｌｉｍｉｔｅｄ Ａａｖ粒子の産生を目的としたベクター
JP2021514659A (ja) 2018-03-06 2021-06-17 ユニバーシティ オブ フロリダ リサーチ ファウンデーション，インコーポレイティド Ａａｖキメラ
JP2022512621A (ja) 2018-10-05 2022-02-07 ボイジャー セラピューティクス インコーポレイテッド Ａａｖ産生タンパク質をコードする操作された核酸コンストラクト

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JP2017506885A (ja) * 2014-01-31 2017-03-16 セクレタリー オブ ステート フォー ヘルスＳｅｃｒｅｔａｒｙ Ｏｆ Ｓｔａｔｅ Ｆｏｒ Ｈｅａｌｔｈ アデノ随伴ウイルスベクターの高力価産生
JP2019513401A (ja) * 2016-04-15 2019-05-30 ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア 新規なａａｖ８変異カプシド及びそれを含有する組成物
WO2019057691A1 (en) 2017-09-19 2019-03-28 Cevec Pharmaceuticals Gmbh INDAVTIBLE AAV GEN GENES
JP2021510496A (ja) 2018-01-19 2021-04-30 オックスフォード ジェネティクス リミテッドＯｘｆｏｒｄ Ｇｅｎｅｔｉｃｓ Ｌｉｍｉｔｅｄ Ａａｖ粒子の産生を目的としたベクター
JP2021514659A (ja) 2018-03-06 2021-06-17 ユニバーシティ オブ フロリダ リサーチ ファウンデーション，インコーポレイティド Ａａｖキメラ
JP2022512621A (ja) 2018-10-05 2022-02-07 ボイジャー セラピューティクス インコーポレイテッド Ａａｖ産生タンパク質をコードする操作された核酸コンストラクト

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OHBA KENJI T, YOSHIHIDE SEHARA, TATSUJI ENOKI, JUNICHI MINENO, KEIYA OZAWA, HIROAKI MIZUKAMI : "Adeno-associated virus vector system controlling capsid expression improves viral quantity and quality", ISCIENCE, vol. 26, no. 4, 21 April 2023 (2023-04-21), pages 106487, XP093095151, DOI: 10.1016/j.isci.2023.106487 *
See also references of EP4644556A1

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120249230A (zh) * 2025-06-05 2025-07-04 鼐济医药科技(杭州)有限公司 一种用于同时生产多种重组腺相关病毒的方法

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EP4644556A1 (en) 2025-11-05
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