Molecular design for adeno-associated virus vector production in recombinant insect cells

• Project/Area Number: 19H02525
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 27040:Biofunction and bioprocess engineering-related
• Research Institution: Kobe University
• Principal Investigator: Yamaji Hideki 神戸大学, 工学研究科, 教授 (40283874)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000); Fiscal Year 2021: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2020: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2019: ¥8,580,000 (Direct Cost: ¥6,600,000、Indirect Cost: ¥1,980,000)
• Keywords: 昆虫細胞 / 組換えタンパク質生産 / アデノ随伴ウイルスベクター / 生物化学工学

Outline of Research at the Start

遺伝子治療は，治療に必要な遺伝子を患者の細胞に導入し，発現させることにより疾患を治療する医療技術である．アデノ随伴ウイルス (AAV) ベクターは安全性が高く，遺伝性疾患を対象とした遺伝子治療において顕著な治療成績を上げている．しかしながら，AAVベクターは作製法が煩雑であるため，高品質なベクターの大量生産は困難となっている．本研究では，昆虫細胞による高品質のAAVベクターの高生産技術を構築するために，AAVベクターの最適な分子設計を検討する．

Outline of Final Research Achievements

A novel production technology for replication-deficient recombinant adeno-associated virus (AAV) vectors that uses insect cells as host cells but does not use baculoviruses was investigated. The genes for AAV regulatory proteins Rep and capsid proteins Cap were introduced into insect cells using plasmid vectors to express Rep and Cap proteins. By using promoters and artificial introns that independently function in insect cells, we demonstrated that AAV vectors can be produced using insect cells as host cells without the use of baculoviruses.

Academic Significance and Societal Importance of the Research Achievements

昆虫細胞－バキュロウイルス系は，AAVベクターの生産性が高い，スケールアップが容易で大量生産に適しているなどの利点を有している．しかしながら，特に遺伝子治療用のウイルスベクターを生産する場合，ベクター粒子とバキュロウイルス粒子の分離が問題となる．本研究では，昆虫細胞で独自に機能するプロモーターと人工イントロンを利用することにより，バキュロウイルスを用いなくとも，昆虫細胞を宿主としてAAVベクターを生産可能であることを明らかにした．宿主として昆虫細胞を用いるもののバキュロウイルスフリーのAAVベクターの生産技術はこれまで報告されおらず，実用的にもきわめて有用である．

Research Products

• [Journal Article] Effects of autophagy inducers on recombinant antibody production in insect cells (2021)
  • Author(s): Ryou Nakanuma, Kyoko Masumi-Koizumi, Yuki Ohmuro-Matsuyama, Tomohisa Katsuda, Hideki Yamaji
  • Journal Title: Cytotechnology Volume: 未定 Issue: 3 Pages: 299-305
  • DOI: 10.1007/s10616-020-00423-6
  • NAID: 120007044365
  • Peer Reviewed
• [Journal Article] Production of an antibody Fab fragment using 2A peptide in insect cells (2020)
  • Author(s): Yu Mizote, Kyoko Masumi-Koizumi, Tomohisa Katsuda, Hideki Yamaji
  • Journal Title: Journal of Bioscience and Bioengneering Volume: 130 Issue: 2 Pages: 205-211
  • DOI: 10.1016/j.jbiosc.2020.03.009
  • NAID: 120007041612
  • Peer Reviewed
• [Journal Article] Production of influenza virus-like particles using recombinant insect cells (2020)
  • Author(s): Takuya Matsuda, Toshikazu Tanijima, Kyoko Masumi-Koizumi, Tomohisa Katsuda, Hideki Yamaji
  • Journal Title: Biochemical Engineering Journal Volume: 163 Pages: 107757-107757
  • DOI: 10.1016/j.bej.2020.107757
  • NAID: 120007041611
  • Peer Reviewed
• [Journal Article] Production of influenza virus proteins using recombinant insect cells (2020)
  • Author(s): Takuya Matsuda, Toshikazu Tanijima, Kyoko Masumi-Koizumi, Tomohisa Katsuda, Hideki Yamaji
  • Journal Title: MATEC Web of Conferences Volume: 333 Pages: 07009-07009
  • DOI: 10.1051/matecconf/202133307009
  • Peer Reviewed / Open Access
• [Journal Article] The effect of telomere sequences on chromosomal translocations (2020)
  • Author(s): Jun Ho Lee, Wataru Tanaka, Noriko Yamano-Adachi, Yuichi Koga, Takeshi Omasa
  • Journal Title: BMC Proceedings 2020 Volume: 14(Suppl 5) Issue: S5 Pages: 16-17
  • DOI: 10.1186/s12919-020-00188-y
  • Peer Reviewed / Open Access
• [Journal Article] Production of influenza virus proteins using recombinant insect cells (2020)
  • Author(s): Takuya Matsuda, Toshikazu Tanijima, Kyoko Masumi-Koizumi, Tomohisa Katsuda, Hideki Yamaji
  • Journal Title: The 18th Asia Pacific Confederation of Chemical Engineering Congress Conference Proceeding Volume: －
  • Peer Reviewed
• [Journal Article] Production of influenza virus-like particles in insect cells (2020)
  • Author(s): Hideki Yamaji, Takuya Matsuda, Toshikazu Tanijima, Kyoko Masumi-Koizumi, Tomohisa Katsuda
  • Journal Title: BMC Proceedings Volume: 未定
• [Journal Article] Production of an antibody Fab fragment using 2A peptide in insect cells (2020)
  • Author(s): Yu Mizote, Kyoko Masumi-Koizumi, Tomohisa Katsuda, Hideki Yamaji
  • Journal Title: Journal of Bioscience and Bioengineering Volume: 未定
  • NAID: 120007041612
  • Peer Reviewed
• [Presentation] 異種タンパク質を提示するインフルエンザウイルス様粒子の昆虫細胞を用いた生産 (2022)
  • Author(s): 市川裕大，松田拓也，Prihardi Kahar，勝田知尚，山地秀樹
  • Organizer: 化学工学会第87年会
• [Presentation] ゲノム編集技術を用いた遺伝子組換え昆虫細胞の樹立 (2021)
  • Author(s): 松田拓也，戸上真也，増見恭子，Prihardi Kahar，勝田知尚，山地秀樹
  • Organizer: 第34回日本動物細胞工学会2021年度大会 (JAACT2021)
• [Presentation] 遺伝子組換え昆虫細胞樹立へのゲノム編集技術の応用 (2021)
  • Author(s): 松田拓也，戸上真也，増見恭子，Prihardi Kahar，勝田知尚，山地秀樹
  • Organizer: 化学工学会第52回秋季大会
• [Presentation] 昆虫細胞を用いたインフルエンザウイルス様粒子の生産 (2020)
  • Author(s): 松田拓也，増見恭子，勝田知尚，山地秀樹
  • Organizer: 化学工学会第51回秋季大会
• [Presentation] Production of influenza virus-like particles using recombinant insect cells (2020)
  • Author(s): Takuya Matsuda, Toshikazu Tanijima, Akito Hirose, Kyoko Masumi-Koizumi, Tomohisa Katsuda, Hideki Yamaji
  • Organizer: The 33rd Annual and International Meeting of the Japanese Association for Animal Cell Technology (JAACT2020 Fuchu)
  • Int'l Joint Research
• [Presentation] Production of influenza virus-like particles in insect cells (2019)
  • Author(s): Hideki Yamaji, Takuya Matsuda, Toshikazu Tanijima, Kyoko Masumi-Koizumi, Tomohisa Katsuda
  • Organizer: 26th ESACT Meeting (ESACT 2019)
  • Int'l Joint Research
• [Presentation] 組換え昆虫細胞によるインフルエンザウイルス抗原タンパク質の生産 (2019)
  • Author(s): 松田拓也，増見恭子，勝田知尚，山地秀樹
  • Organizer: 第32回日本動物細胞工学会2019年度大会 (JAACT2019)
• [Presentation] Production of influenza virus proteins in recombinant insect cells (2019)
  • Author(s): Takuya Matsuda, Toshikazu Tanijima, Kyoko Masumi-Koizumi, Tomohisa Katsuda, Hideki Yamaji
  • Organizer: 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019)
  • Int'l Joint Research
• [Book] バイオリアクターのスケールアップと物質生産事例集 (2021)
  • Author(s): 山地秀樹
  • Publisher: 技術情報協会
  • ISBN: 9784861048302
• [Book] 医薬品モダリティの特許戦略と技術開発動向 (2019)
  • Author(s): 山地秀樹
  • Publisher: 技術情報協会
  • ISBN: 9784861047497