適正製造規範に適合する簡易精製が可能な次世代AAVベクターの開発

2015 Fiscal Year Annual Research Report

• Project/Area Number: 25460407
• Research Institution: Nippon Medical School
• Principal Investigator: 平井 幸彦 日本医科大学, 医学部, 講師 (10089617)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: AAVベクター精製法 / 遺伝子細胞治療 / 適正製造規範準拠 / rAAV 9 vector

Outline of Annual Research Achievements

第１世代の2型や5型AAVのキャプシドのheparinやムチンに対する親和性に基づいて、第２世代のキャプシドを種々に混合した新たなAAVベクターを作成し、そのアフィニティークロマトを用いて簡易精製する方法の開発を申請した。しかし、実験を進める過程で、type 1 およびtype 9 AAVベクターの各々を、適正製造規範(GMP)への準拠が可能な簡易精製法を確立できる可能性を見出した。そこで、本年は高価なアフィニティークロマト用カラムの必要が無く、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の確立を試みた。
無血清培養液下のHEK293 EB細胞へ３つのプラスミドを遺伝子導入後、９型AAVベクターを細胞培養液中に分泌させた。細胞培養液を限外ろ過して、低分子量のタンパク質を除去しながら濃縮・精製し、牛胎児血清や細胞由来のタンパク質の混入の少ない粗AAVベクター分画を得た。1/3飽和硫安処理により不要な共存蛋白質を遠心除去後、1/2飽和硫安処理により、AAV vector を沈殿させた。沈渣を3.3mM MES-HEPES-NaOAc緩衝液pH8.0にて、電気伝導度が7.3 ｍS/cm まで希釈後、HiPrep Q XL16/10 に負荷して、流出液としてベクターを回収した。SDS-PAGEの蛋白質染色で、ほぼVP1 ,VP2 ,VP3 の3本のバンドとなり、電子顕微鏡像によって、約97%の粒子がウイルス・ゲノムを有しており、実際の遺伝子細胞治療に使用可能な高力価・高純度であることが示された。最終的に　5 x 10E+9 のHEK293 EB細胞より、1.5 x 10E+15 viral genome の精製AAVベクターをdown-stream processingによって得ることが出来た。現在、Molecular Therapy に投稿中である。

Research Products

• [Journal Article] Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1). (2016)
  • Author(s): Tomono, T., Hirai, Y., Okada, H., Adachi, K., Ishii, A., Shimada, T., Onodera, M., Tamaoka, A., Okada T.
  • Journal Title: Molecular Therapy -Methods & Clinical Development Volume: 3 Pages: 15058
  • DOI: 10.1038/mtm.2015.58
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Treatment of hypophosphatasia by muscle-directed expression of bone-targeted alkaline phosphatase via self-complementary AAV8 vector. (2016)
  • Author(s): Nakamura-Takahashi,A., Miyake,K., Watanabe, A., Hirai, Y., Iijima, O., Miyake, N., Adachi K.,, Nitahara-Kasahara, Y., Kinoshita, H., Noguchi, T., Abe, S., Narisawa, S., Millán, J. L., Shimada, T.,Okada, T.
  • Journal Title: Molecular Therapy- Methods & Clinical Development Volume: 3 Pages: 15059
  • DOI: 10.1038/mtm.2015.59
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Enzyme replacement in the CSF to treat metachromatic leukodystrophy in mouse model using single intracerebroventricular injection of self-complementary AAV1 vector. (2015)
  • Author(s): Hironaka, K., Yamazaki, Y., Hirai, Y., Yamamoto. M., Miyake, N., Miyake, K., Okada, T., Morita, A., Shimada, T.
  • Journal Title: SCIENTIFIC REPORTS Volume: 5 Pages: 13104
  • DOI: 10.1038/srep13104
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Ultracentrifugation-free Chromatography-mediated Large-scale Purification of Recombinant Adeno-Associated Virus Serotype 1 (rAAV1) and rAAV9 from the Serum-free Culture Supernatant (2016)
  • Author(s): Tomono, T., Hirai, Y., Okada, H., Adachi、K., Ishii, A., Shimada, T., Onodera, M., Tamaoka, A., Okada T
  • Organizer: The 19th Annual Meeting of the American Society of Gene & Cell Therapy
  • Place of Presentation: Washington, DC, USA.
  • Year and Date: 2016-05-04 – 2016-05-07
  • Int'l Joint Research
• [Presentation] The large-scale purification of rAAV1 from the serum-free cultured medium by ion-exchange and gel-filtration chromatography-steps with ultracentrifugation-free technique. (2015)
  • Author(s): Tomono, T., Hirai, Y., Okada, H., Adachi, K., Chiyo, T., Ishii, A., Shimada, T., Onodera, M., Tamaoka, A., Okada T.
  • Organizer: The 21st Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: Osaka, JAPAN
  • Year and Date: 2015-07-24 – 2015-07-26
  • Int'l Joint Research
• [Presentation] Gene therapy to rescue lethal hypophosphatasia model mice by adeno-associated virus-mediated muscle transduction of bone-targeted alkaline phosphatase. (2015)
  • Author(s): Nakamura-Takahashi,A., Miyake K., Watanabe, A., Hirai, Y., Miyake, N., Iijima, O., Adachi K., Kinoshita, H., Noguchi, T., Abe, S.,Shimada, T.,Okada, T.
  • Organizer: The 21st Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: Osaka, JAPAN
  • Year and Date: 2015-07-24 – 2015-07-26
  • Int'l Joint Research
• [Presentation] Efficient Scalable Purification of rAAV1 Using Ion-Exchange and Gel-Filtration Chromatography To Avoid Ultracentrifugation Procedure. (2015)
  • Author(s): Tomono, T., Hirai, Y., Okada, H., Chiyo, T., Shimada, T., Onodera, M.., Okada T.
  • Organizer: The 18th Annual Meeting of the American Society of Gene & Cell Therapy
  • Place of Presentation: New Orleans, Louisiana, USA.
  • Year and Date: 2015-05-13 – 2015-05-16
  • Int'l Joint Research
• [Presentation] Prolonged Survival and Improved Phenotypes of Lethal Hypophosphatasia Model Mice by Adeno-Associated Virus-Mediated Muscle Transduction of Bone-Targeted Alkaline Phosphatase. (2015)
  • Author(s): Nakamura-Takahashi,A., Miyake K., Watanabe, A., Hirai, Y., Miyake, N., Iijima, O., Adachi K., Kinoshita, H., Noguchi, T., Abe, S., Shimada, T., Okada, T.
  • Organizer: The 18th Annual Meeting of the American Society of Gene & Cell Therapy
  • Place of Presentation: New Orleans, Louisiana, USA.
  • Year and Date: 2015-05-13 – 2015-05-16
  • Int'l Joint Research