Purification of rAAV with ultracentrifugation-free technique towards GMP prodction Part 2

• Project/Area Number: 16K08644
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Human genetics
• Research Institution: Nippon Medical School
• Principal Investigator: Hirai Yukihiko 日本医科大学, 大学院医学研究科, 研究生 (10089617)
• Project Period (FY): 2016-10-21 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: AAVベクター / 適正製造規範 / 簡易精製法 / 分析用超遠心 / ハイブリッドベクター / 分析用超遠心機 / AAV ベクター / AAVベクター作成 / xeno-free system

Outline of Final Research Achievements

A high quality AAV9 vector purification method was established by chromatographic techniques only. The virus genome sequence and genome structure were also independent, and AAV9 vector with a total titer of 10E + 14 virus genome or more could be prepared. The ratio of the hollow particles was calculated from electron microscopic observation, and it was shown that the purification method had reproducibility as high purity as 5% or less. Analysis of particles containing a full-length genome by analytical ultracentrifugation results in 38%, suggesting that further refinement of the preparation and purification method is necessary.These results was published in Mol Ther Methods Clin Dev. 2018; 11: 180-190.

Academic Significance and Societal Importance of the Research Achievements

標準作業手順上の煩雑さを含む超遠心分離を含まないtype 9 AAVベクターの作成・精製のdown-stream processingを確立した。ウイルスゲノム配列（GOI）・ゲノム構造にも非依存性で、10E+14 ウイルスゲノム 以上の総力価を大学研究室設備で調製できた。電子顕微鏡観察からの中空粒子の混入率はどれも5%以下と、高純度に再現性をもつ精製方法であった。しかし、分析用超遠心分離法による全長ゲノムを含む粒子の解析では、アフィニティークロマトを用いない本法でも38% となり、高い遺伝子導入効率を有するAAVベクターの作成・精製にはの更なる改良が必要であることを示唆した。

Research Products

• [Journal Article] Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9 (2018)
  • Author(s): Tomono Ｔ, Hirai Y, Okada H, Miyagawa Y, Adachi 1, Sakamoto S, Kawano Y, Chono H, Mineno J, Ishii A, Shimada T, Onodera M, Tamaoka A, Okada T.
  • Journal Title: Mol Ther Methods Clin Dev Volume: １１ Pages: 180-190
  • DOI: 10.1016/j.omtm.2018.10.015
  • NAID: 120007128300
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation. (2016)
  • Author(s): Igarashi, T., Miyake, K., Kobayashi, M., Kameya, S., Fujimoto, C., Nakamoto, K., Takahashi, H., Igarashi, T., Miyake, N., Iijima, O., Hirai,Y., Shimada, T., Okada, T., Takahashi, H.
  • Journal Title: Molecular Vision Volume: 22 Pages: 816-826
  • Peer Reviewed / Open Access
• [Journal Article] Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1). (2016)
  • Author(s): Taro Tomono, Yukihiko Hirai, Hironori Okada, Kumi Adachi, Akiko Ishii, Takashi Shimada, Masafumi Onodera, Akira Tamaoka, and Takashi Okada
  • Journal Title: Molecular Therapy - Methods & Clinical Development Volume: 3 Pages: 15058-15058
  • DOI: 10.1038/mtm.2015.58
  • NAID: 120007128523
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Refinements of rAAV8 purification protocol with chromatography technology (2018)
  • Author(s): 3）Taro Tomono, Yukihiko Hirai, Hironori Okada, Yoshitaka Miyagawa, Kumi Adachi, Akiko Ishii, Takashi Shimada, Akira Tamaoka, Takashi Okada.
  • Organizer: The ASGCT 21st annual meeting
  • Int'l Joint Research
• [Presentation] クロマトグラフィー技術を用いた8型アデノ随伴ウイルスベクター(rAAV8) の精製 (2017)
  • Author(s): 伴野太郎、増田千明、宮川世志幸、岡田浩典、平井幸彦、石井亜紀子、玉岡晃、岡田尚巳
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] イオン交換カラムクロマトグラフィー技術による組換え5型アデノ随伴ウイルス精製法の開発 (2017)
  • Author(s): 増田千明、伴野太郎、宮川世志幸、岡田浩典、平井幸彦、岡田尚巳
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] rAAV-8 production by using HEK293 derivatives, xeno-free media and flow electroporation-based transfection (2017)
  • Author(s): Hironori Okada, Taro Tomono,, Kumi Adachi, Yukihiko Hirai, Masaaki Ouchi, Yuko Utsunomiya, Hideto Chono, Junichi Mineno, Takashi Okada
  • Organizer: American Society of Gene and Cell Therapy 20th Annual Meeting
  • Int'l Joint Research
• [Presentation] Ultracentrifugation-free Chromatography-mediated Purification of Recombinant Adeno-Associated Virus Serotype 9 (rAAV9) (2017)
  • Author(s): Taro Tomono, Yukihiko Hirai, Hironori Okada, Yoshitaka Miyagawa, Kumi Adachi, Shuhei Sakamoto, Yasuhiro Kawano, Hideto Chono, Junichi Mineno, Akiko Ishii, Takashi Shimada, Masafumi Onodera, Akira Tamaoka, Takashi Okada
  • Organizer: American Society of Gene and Cell Therapy 20th Annual Meeting
  • Int'l Joint Research
• [Presentation] Development of flow electroporation-based suspension-adapted HEK293 transfection system toward rAAV production in GMP (2017)
  • Author(s): Hironori Okada, Taro Tomono, Kumi Adachi, Yukihiko Hirai, Masaaki Ouchi, Yuko Utsunomiya, Hideto Chono, Junichi Mineno, Takashi Okada
  • Organizer: The 23rd Annual Meeting of Japan Society of Gene and Cell Therapy
  • Int'l Joint Research
• [Presentation] Purification of recombinant adeno-associated virus with only chromatography technique (2017)
  • Author(s): Taro Tomono, Chiaki Masuda, Yukihiko Hirai, Hironori Okada, Yoshitaka Miyagawa, Kumi Adachi, Shuhei Sakamoto, Yasuhiro Kawano, Hideto Chono, Junichi Mineno, Akiko Ishii, Takashi Shimada, Masafumi Onodera, Akira Tamaoka, Takashi Okada
  • Organizer: The 23rd Annual Meeting of Japan Society of Gene and Cell Therapy
  • Int'l Joint Research
• [Patent(Industrial Property Rights)] 筋委縮性側索硬化症を含む神経疾患の予防剤及び／又は治療剤 (2018)
  • Inventor(s): 望月秀樹、長野清一、佐々木勉、鐘其静と、岡田尚巳、平井幸彦
  • Industrial Property Rights Holder: 望月秀樹、長野清一、佐々木勉、鐘其静と、岡田尚巳、平井幸彦
  • Industrial Property Rights Type: 特許
  • Filing Date: 2018