Development of rAAV-mediated brain transduction with transient ultrasound-mediated modulation of the blood-brain interface in adult common marmoset toward modeling epilepsy

• Project/Area Number: 26430102
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Laboratory animal science
• Research Institution: Nippon Medical School
• Principal Investigator: Okada Hironori 日本医科大学, 大学院医学研究科, 研究員 (80416271)
• Co-Investigator(Renkei-kenkyūsha): OKADA Takashi 日本医科大学, 大学院医学研究科, 大学院教授 (00326828)
• Research Collaborator: TAKEDA Shinichi 国立精神・神経医療研究センター, 神経研究所, 所長 (90171644) ; KINOH Hiromi 日本医科大学, 医学部, 助教 (60532728) ; KASAHARA Yuko 日本医科大学, 大学院医学研究科, 助教 (90391911)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2014: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: AAVベクター / てんかん / マーモセット / 超音波 / 血液脳関門 / 疾患モデル / 成体

Outline of Final Research Achievements

The common marmoset could provide an appropriate model for neuromuscular diseases because of its higher brain function and physiological resemblance to human. An adeno-associated virus (AAV) vector has great advantages to induce pathology in the aged animals. However, fully maturated blood-brain barrier (BBB) significantly limits passive rAAV transport from circulation to the brain. We investigated microbubble(MB)-assisted transcranial ultrasound irradiation (TCUI) BBB opening technology in the adult marmoset. TCUI immediately after bolus injection of MB resulted in the BBB opening in blood vessels in some areas including the hippocampus, and it seemed to be repaired by at least 2 days after TCUI. In parallel, we constructed self-complementary AAV2 vector proviral plasmids expressing dominant negative mutants of sodium ion channel genes (SCN1A or SCN2A), which causes Dravet syndrome. The vector genomes could be packaged into the AAV9 capsid and purified by conventional method.

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] Intra-Amniotic rAAV-Mediated Microdystrophin Gene Transfer Improves Canine X-Linked Muscular Dystrophy and May Induce Immune Tolerance. (2015)
  • Author(s): Hiromi Hayashita-Kinoh, Naoko Yugeta, Hironori Okada, Yuko Nitahara-Kasahara, Tomoko Chiyo, Takashi Okada and Shin’ichi Takeda
  • Journal Title: Mol. Ther. Volume: 4 Issue: 4 Pages: 627-637
  • DOI: 10.1038/mt.2015.5
  • Peer Reviewed / Open Access
• [Journal Article] Dystrophic mdx mice develop severe cardiac and respiratory dysfunction following genetic ablation of the anti-inflammatory cytokine IL-10. (2015)
  • Author(s): Yuko Nitahara-Kasahara, Hiromi Hayashita-Kinoh, Tomoko Chiyo, Akiyo Nishiyama, Hironori Okada, Shin’ichi Takeda and Takashi Okada.
  • Journal Title: Hum Molecular Genetics Volume: 23（15） Issue: 15 Pages: 3990-4000
  • DOI: 10.1093/hmg/ddu113
  • Peer Reviewed / Open Access
• [Presentation] Feasibility of large-scale rAAV production from suspension HEK293 cell line using flow electroporation system (2016)
  • Author(s): Okada H., Tomono T., Adachi K., Hirai Y., Okada T
  • Organizer: The 22nd Annual Meeting of Japan Society of Gene and Cell Therapy
  • Place of Presentation: 東京
  • Year and Date: 2016-07-28
• [Presentation] Cell therapeutic approach using dental pulp stromal cells for Duchenne muscular dystrophy (2016)
  • Author(s): Nitahara-Kasahara Y., Kuraoka M., Hayashita-Kinoh H., Nakamura-Takahashi A., Masuda C., Imagawa K., Tachibana K., Takeda S., Okada T
  • Organizer: The 22nd Annual Meeting of Japan Society of Gene and Cell Therapy
  • Place of Presentation: 東京
  • Year and Date: 2016-07-28
• [Presentation] Systemic administration of rAAV9-microdystrophin with MSCs pre-treatment improves transgene expression and phenotype in CXMDJ (2016)
  • Author(s): Hayashita-Kinoh H., Nitahara-Kasahara Y., Kuraoka M., Okada H., Imagawa K., Tachibana K., Takeda S., Okada T
  • Organizer: The 22nd Annual Meeting of Japan Society of Gene and Cell Therapy
  • Place of Presentation: 東京
  • Year and Date: 2016-07-28
• [Presentation] Simple and effective ultracentrifugation-free large-scale purification of recombinant adeno-associated virus serotype 9 (rAAV9) (2016)
  • Author(s): Tomono T., Hirai Y., Okada H., Adachi K., Sakamoto S., Kawano Y., Chono H., Mineno J., Ishii A., Shimada T., Onodera M., Tamaoka A., Okada T
  • Organizer: The 22nd Annual Meeting of Japan Society of Gene and Cell Therapy
  • Place of Presentation: 東京
  • Year and Date: 2016-07-28
• [Presentation] Effective microdystrophin expression in non-human primate muscle with AAV type 8 vectors under immune suppression (2016)
  • Author(s): Ishii A., Okada H., Hayashita-Kinoh H., Shin J. H., Okada T., Takeda S
  • Organizer: The 22nd Annual Meeting of Japan Society of Gene and Cell Therapy
  • Place of Presentation: 東京
  • Year and Date: 2016-07-28
• [Presentation] Transient Ultrasound-Mediated Microbubble-Assisted Modulation of Blood-Brain Interface in Adult Common Marmoset to Improve rAAV-Mediated Brain Transduction (2016)
  • Author(s): Okada H, Ishibashi H, Masuda C, Hayashita-Kinoh H, Chiyo T, Nitahara-Kasahara Y, Endo-Takahashi Y, Kato K, Negishi Y, Takeda S, Okada T
  • Organizer: American society of gene & cell therapy 19th Annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2016-05-04
  • Int'l Joint Research
• [Presentation] Improved Transduction of Canine X-Linked Muscular Dystrophy with rAAV9-Microdystrophin by Introducing Immune Tolerance (2016)
  • Author(s): Hayashita-Kinoh H, Okada H, N. Kasahara Y, Chiyo T, Imagawa K, Tachibana K, Takeda S, Okada T
  • Organizer: American society of gene & cell therapy 19th Annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2016-05-04
  • Int'l Joint Research
• [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: American society of gene & cell therapy 19th Annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2016-05-04
  • Int'l Joint Research
• [Presentation] Ultrasound-mediated transient modulation of blood-brain interface in adult common marmosetto induce brain pathology with rAAV. (2015)
  • Author(s): Okada H, Ishibashi H, Masuda C, Hayashita-Kinoh H, Chiyo T, Nitahara-Kasahara Y, Endo-Takahashi Y, Kato K, Negishi Y, Takeda S, Okada T
  • Organizer: The 21st Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 大阪
  • Year and Date: 2015-07-24
• [Presentation] Mesenchymal stromal cells ameliorate progressive phenotype of Duchenne muscular dystrophy in dog. (2015)
  • Author(s): Kasahara Y, Kinoh H, Kuraoka M, Chiyo T, Okada H, Tsumita N, Imagawa K, Tachibana K, Takeda S, Okada T
  • Organizer: The 21st Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 大阪
  • Year and Date: 2015-07-24
• [Presentation] Feasible gene transduction in non-human primate muscle with reconbinantAAV following immunomodulation. (2015)
  • Author(s): Ishii A, Okada H, Hayashita-Kinoh H, Shin J, Okada T, Takeda S
  • Organizer: The 21st Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 大阪
  • Year and Date: 2015-07-24
• [Presentation] Improved transduction of canine X-linked muscular dystrophy with rAAV9- microdystrophin by MSCs pretreatment Improved transduction of canine X-linked muscular dystrophy with rAAV9- microdystrophin by MSCs pretreatment. (2015)
  • Author(s): Hayashita-Kinoh H, Nitahara-Kasahara Y, Okada H, Chiyo T, Imagawa K, Tachibana K, Takeda S, Okada T
  • Organizer: The 21st Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 大阪
  • Year and Date: 2015-07-24
• [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: 大阪
  • Year and Date: 2015-07-24
• [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: 18th Annual Meeting of American Society of Gene & Cell Therapy
  • Place of Presentation: New Orleans, LA, USA
  • Year and Date: 2015-05-13
  • Int'l Joint Research
• [Presentation] Blood-Brain Interface Opening By Ultrasound in Adult Common Marmoset To Induce Brain Pathology With rAAV. (2015)
  • Author(s): Okada H, Ishibashi H, Masuda C, Hayashita-Kinoh H, Chiyo T, Nitahara-Kasahara Y, Endo-Takahashi Y, Kato K, Negishi Y, Takeda S, Okada T
  • Organizer: 18th Annual Meeting of American Society of Gene & Cell Therapy
  • Place of Presentation: New Orleans, LA, USA
  • Year and Date: 2015-05-13
  • Int'l Joint Research
• [Presentation] Improved Transduction of Canine X-Linked Muscular Dystrophy With rAAV9-Microdystrophin By Using MSCs Pretreatment. (2015)
  • Author(s): Hayashita-Kinoh H, Okada H, Nitahara-Kasahara Y, Chiyo T, Imagawa K, Tachibana K, Takeda S, Okada T
  • Organizer: 18th Annual Meeting of American Society of Gene & Cell Therapy
  • Place of Presentation: New Orleans, LA, USA
  • Year and Date: 2015-05-13
  • Int'l Joint Research
• [Presentation] Mesenchymal Stromal Cells Can Ameliorate the Progressive Phenotype of Dog With Duchenne Muscular Dystrophy. (2015)
  • Author(s): Nitahara-Kasahara Y, Hayashita-Kinoh H, Kuraoka M, Chiyo T, Okada H, Tsumita N, Imagawa K, Tachibana K, Takeda S, Okada T
  • Organizer: 18th Annual Meeting of American Society of Gene & Cell Therapy
  • Place of Presentation: New Orleans, LA, USA
  • Year and Date: 2015-05-13
  • Int'l Joint Research
• [Presentation] Engraftment of mesenchymal stromal cells is effectively associated by IL-10 in skeletal muscle. (2014)
  • Author(s): Nitahara-Kasahara Y, Hayashita-Kinoh H, Tsumita N, Chiyo T, Okada H, Takeda S, Okada T
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 東京
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] Immune tolerance induction of canine X-linked muscular dystrophy with fetal rAAV-microdystrophin transduction. (2014)
  • Author(s): Hayashita-Kinoh H, Okada H, Nitahara-Kasahara Y, Chiyo T, Yugeta N, Okada T, Takeda S
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 東京
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] rAAV1 and 8-mediated induction of local OPMD histopathology in common marmotset. (2014)
  • Author(s): Okada H, Ishibashi H, Hayashita-Kinoh H, Chiyo T, Nitahara-Kasahara Y, Takeda S, Okada T
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 東京
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] IL-10強制発現による機能強化型MSCsの作製と生存解析. (2014)
  • Author(s): 笠原優子, 喜納裕美, 千代智子, 岡田浩典, 武田伸一, 岡田尚巳
  • Organizer: 第35回日本炎症・再生医学会
  • Place of Presentation: 沖縄
  • Year and Date: 2014-07-01 – 2014-07-04
• [Presentation] Induction of Local OPMD Histopathology in Common Marmoset By rAAV1 and 8-Mediated Transduction (2014)
  • Author(s): Okada H, Ishibashi H, Hayashita-Kinoh H, Chiyo T, Masuda C, Nitahara-Kasahara Y, Takeda S, Okada T
  • Organizer: American society of gene & cell therapy 17th Annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2014-05-21 – 2014-05-24
• [Presentation] Skeletal Muscle Engraftment of Mesenchymal Stromal Cells Is Augmented By IL-10 (2014)
  • Author(s): Nitahara-Kasahara Y, Hayashita-Kinoh H, Chiyo T, Okada H, Takeda S, Okada T
  • Organizer: American society of gene & cell therapy 17th Annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2014-05-21 – 2014-05-24
• [Presentation] Immune Tolerance Induction in Canine X-Linked Muscular Dystrophy With rAAV9-Microdystrophin Transduction (2014)
  • Author(s): Hayashita-Kinoh H, Nitahara-Kasahara Y, Okada H, Chiyo T, Yugeta N, Okada T, Takeda S
  • Organizer: American society of gene & cell therapy 17th Annual meeting
  • Place of Presentation: Washington DC, USA
  • Year and Date: 2014-05-21 – 2014-05-24
• [Book] 血液フロンティア: ゲノム編集技術を用いる遺伝子治療 (2015)
  • Author(s): 岡田 浩典、伴野 太郎、岡田 尚巳
  • Total Pages: 16
  • Publisher: 医薬ジャーナル社
• [Book] Pharma Medica: 遺伝子導入用ウイルスベクターの特徴と作成法 (2015)
  • Author(s): 伴野 太郎、岡田 浩典、岡田 尚巳
  • Total Pages: 8
  • Publisher: メディカルレビュー社