Purification of rAAV-1 and -9 with ultracentrifugation-free technique towards GMP production.

• Project/Area Number: 25460407
• 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, 医学部, 講師 (10089617)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2015: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2013: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: AAV ベクター精製法 / 適正製造規範準拠 / down-stream processing / AAV type 1 vector / AAV type 9 vector / AAVベクター精製法 / 遺伝子細胞治療 / rAAV 9 vector / down-steam processing

Outline of Final Research Achievements

The current production of rAAV from the transfected cell lysate and purification based on CsCl or iodixanol density ultracentrifugation are not suitable for large-scale processing. Although rAAV1 and rAAV9 are promising therapeutic vectors for genetic neuromuscular disorders, the large-scale purification method for those vectors has not yet been established. In this study, we elaborate the novel chromatography-mediated methods for purification of rAAV1 and rAAV9 from the serum-free culture supernatant with ultracentrifugation-free technique towards large-scale and GMP production.

Research Products

• [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
• [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: 15059-15059
  • 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: Sci Rep. Volume: 18 Issue: 1 Pages: 1-12
  • DOI: 10.1038/srep13104
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Intrathecal AAV Serotype 9-mediated Delivery of shRNA Against TRPV1 Attenuates Thermal Hyperalgesia in a Mouse Model of Peripheral Nerve Injury. (2014)
  • Author(s): Hirai T, Enomoto M, Kaburagi H, Sotome S, Yoshida-Tanaka K, Ukegawa M, Kuwahara H, Yamamoto M, Tajiri M, Miyata H, Hirai Y, Tominaga M, Shinomiya K, Mizusawa H, Okawa A, Yokota T
  • Journal Title: Mol Ther Volume: 22 Issue: 2 Pages: 409-19
  • DOI: 10.1038/mt.2013.247
  • Peer Reviewed
• [Journal Article] Targeted gene transfer into ependymal cells through intraventricular injection of AAV1 vector and long-term enzyme replacement via the CSF. (2014)
  • Author(s): Yamazaki Y, Hirai Y, Miyake K, Shimada T.
  • Journal Title: Scientific Reports Volume: 4 Issue: 1 Pages: 5506-5506
  • DOI: 10.1038/srep05506
  • Peer Reviewed / Open Access
• [Journal Article] Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice. (2013)
  • Author(s): Akira Machida, Hiroya Kuwahara, Azat Mayra, Takayuki Kubodera, Takashi Hirai, Fumiko Sunaga, Mio Tajiri, Yukihiko Hirai, Takashi Shimada, Hidehiro Mizusawa, Takanori Yokota.
  • Journal Title: Mol Pain. Volume: 9 Pages: 36-45
  • DOI: 10.1186/1744-8069-9-36
  • Peer Reviewed
• [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
  • 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
  • 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
  • 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
  • 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
  • Int'l Joint Research
• [Presentation] Tyrosine-mutated AAV2 mediated BDNF rescued inner retina in rat retinal ischemic injury mode. (2015)
  • Author(s): Igarash T, Miyake K, Kobayashi M, Miyake N, Iijima O, Nakamoto K, Hirai Y, Shimada T, Okada T, Takahashi T.
  • Organizer: The Association for Research in Vision and Ophthalmology, Inc. (ARVO) Asia-ARVO 2015
  • Place of Presentation: Yokohama, Japan
  • Year and Date: 2015-02-16 – 2015-02-19
• [Presentation] Rescue of lethal hypophosphatasia model mice by adeno-associated virus mediated muscle specific expression of bone targeted alkaline phosphatase. (2014)
  • Author(s): Nakamura A, Iijima O, Miyake K, Watanabe A, Hirai Y, Kinoshita H, Noguchi T, Abe S, Okada T, Shimada T.
  • Organizer: The 64th Annual Meeting of the American Society of Human Genetics
  • Place of Presentation: San Diego, California,USA
  • Year and Date: 2014-10-18 – 2014-10-22
• [Presentation] Tyrosine-mutated AAV2 mediated BDNF rescued inner retina in rat retinal ischemic injury model (2014)
  • Author(s): Igarashi T, Miyake K., Kobayashi M, Takahashi K., Miyake N., lijima 0., Nakamoto K., Hirai Y, Shimada T, Okada T, Takahashi H
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy (JSGT2014)
  • Place of Presentation: Tokyo, JAPAN
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] Production and purification of AAV1 and AAV9 vectors without using ultracentrifugation (2014)
  • Author(s): Tomono T, Hirai Y., Shimada T., Onodera M., Okada T.
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy (JSGT2014)
  • Place of Presentation: Tokyo, JAPAN
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] Improvement of hypophosphatasia model mice by muscle specific expression of bone targeted alkaline phosphatase using self-complementary AAV8 vector. (2014)
  • Author(s): Nakarmura A, Iijima 0, Miyake K., Watanabe A., Hirai Y, Kinoshita H, Noguchi T, Abe S., Okada T, Shimada T
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy (JSGT2014)
  • Place of Presentation: Tokyo, JAPAN
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] Long-term enzyme supplementation into the CSF to treat metachromatic leukodystrophy by intraventricular injection of AAV1 vector (2014)
  • Author(s): Yamazaki Y., Hironaka K, Miyake N., Hirai Y., Miyake K., Okada T., Shimada T.
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy (JSGT2014)
  • Place of Presentation: Tokyo, JAPAN
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] Efficient Protection of Retina By Tyrosine-Mutated Self-Complementary AAV2 Vector Encoding BDNF in a Rat Retinal Ischemic Injury Model (2014)
  • Author(s): Igarashi T, Miyake K, Kobayashi, Takahashi K, Miyake N, Iijima O, Nakamoto K, Hirai Y, Shimada T, Takahashi H.
  • Organizer: The 17th Annual Meeting of the American Society of Gene & Cell Therapy
  • Place of Presentation: Washington, DC、USA
  • Year and Date: 2014-05-21 – 2014-05-24
• [Presentation] Targeted Gene Transfer into the Ependymal Cells and Long－Term Enzyme Replacement in the CSF by Intraventricular Injection of AAV Vector Serotype 1 (2013)
  • Author(s): Yoshiyuki Yamazaki, Kohei Hironaka, Yukihiko Hirai, Koichi Miyake, Takashi Shimada
  • Organizer: The American Society of Gene & Cell Therapy
  • Place of Presentation: Salt Lake City, Utah, USA .