Development of gene therapy against neurodegenerative diseases using CRISPR/Cas9-based genome editing

• Project/Area Number: 16K15477
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Neurology
• Research Institution: Gunma University
• Principal Investigator: Hirai Hirokazu 群馬大学, 大学院医学系研究科, 教授 (70291086)
• Co-Investigator(Renkei-kenkyūsha): HOSOI Nobutake 群馬大学, 大学院医学系研究科, 講師 (90543570) ; KONNO Ayumu 群馬大学, 大学院医学系研究科, 講師 (40509048)
• Research Collaborator: MATSUZAKI Yasunori 群馬大学, 大学院医学系研究科, 助教 (50738200)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2017: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000); Fiscal Year 2016: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
• Keywords: 小脳 / プルキンエ細胞 / 細胞種特異的プロモーター / CRISPR/Cas9 / ゲノム編集 / AAV / ウイルスベクター / 遺伝子治療 / SCA1 / AAVベクター / アデノ随伴ウイルスベクター / 脊髄小脳失調症 / 遺伝子 / 再生医学 / 神経科学 / 脳神経疾患

Outline of Final Research Achievements

In this research, we aimed to establish a technique that allows elimination of a disease-causing gene from specific cell populations in the brain, using adeno-associated virus (AAV) vectors in combination with CRISPR/Cas9 system. Initially, we characterized neuron-specific, astrocyte-specific and cerebellar Purkinje cell-specific promoters available for AAV vector-mediated transgene expression. The results were published in 3 papers. Then, using the AAV vectors, we examined whether a gene responsible for developmental defects of the brain could be knocked out from Purkinje cells by CRISPR/Cas9 system. The results showed successful removal of the gene from around two thirds of Purkinje cells.

Research Products

• [Int'l Joint Research] Oxford University(United Kingdom)
• [Int'l Joint Research] Trustees of Dartmouth College/University of Tennessee/Max Planck Florida Institute(米国)
• [Journal Article] Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain (2018)
  • Author(s): Matsuzaki Yasunori、Konno Ayumu、Mochizuki Ryuta、Shinohara Yoichiro、Nitta Keisuke、Okada Yukihiro、Hirai Hirokazu
  • Journal Title: Neuroscience Letters Volume: 665 Pages: 182-188
  • DOI: 10.1016/j.neulet.2017.11.049
  • Peer Reviewed
• [Journal Article] Elavl3 is essential for the maintenance of Purkinje neuron axons (2018)
  • Author(s): Ogawa Yuki、Kakumoto Kyoko、Yoshida Tetsu、Kuwako Ken-ichiro、Miyazaki Taisuke、Yamaguchi Junji、Konno Ayumu、Hata Junichi、Uchiyama Yasuo、Hirai Hirokazu、Watanabe Masahiko、Darnell Robert B.、Okano Hideyuki、Okano Hirotaka James
  • Journal Title: Sci Rep. Volume: 8 Issue: 1 Pages: 2722-2722
  • DOI: 10.1038/s41598-018-21130-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Viral Vector-Based Evaluation of Regulatory Regions in the Neuron-Specific Enolase (NSE) Promoter in Mouse Cerebellum In Vivo (2017)
  • Author(s): Shinohara Yoichiro、Ohtani Toshinori、Konno Ayumu、Hirai Hirokazu
  • Journal Title: The Cerebellum Volume: 16 Issue: 5-6 Pages: 913-922
  • DOI: 10.1007/s12311-017-0866-5
  • Peer Reviewed
• [Journal Article] Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging (2017)
  • Author(s): Harada Kazuki、Ito Motoki、Wang Xiaowen、Tanaka Mika、Wongso Devina、Konno Ayumu、Hirai Hirokazu、Hirase Hajime、Tsuboi Takashi、Kitaguchi Tetsuya
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 7351-7351
  • DOI: 10.1038/s41598-017-07820-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Minimal Purkinje Cell-Specific PCP2/L7 Promoter Virally Available for Rodents and Non-human Primates (2017)
  • Author(s): Nitta Keisuke、Matsuzaki Yasunori、Konno Ayumu、Hirai Hirokazu
  • Journal Title: Molecular Therapy - Methods & Clinical Development Volume: 6 Pages: 159-170
  • DOI: 10.1016/j.omtm.2017.07.006
  • Peer Reviewed / Open Access
• [Journal Article] Regulatory connection between the expression level of classical protein kinase C and pruning of climbing fibers from cerebellar Purkinje cells (2017)
  • Author(s): Takahashi Nobutaka、Shuvaev Anton N.、Konno Ayumu、Matsuzaki Yasunori、Watanave Masashi、Hirai Hirokazu
  • Journal Title: Journal of Neurochemistry Volume: 143 Issue: 6 Pages: 660-670
  • DOI: 10.1111/jnc.14239
  • Peer Reviewed / Int'l Joint Research
• [Presentation] In vivo evaluation of the shortened-WPRE in the cerebellum (2017)
  • Author(s): Ayumu Konno, Kazuma Hachisu, Hirokazu Hirai
  • Organizer: 第40回日本神経科学大会
• [Presentation] アデノ随伴ウイルスによる遺伝子導入を介したリピート病モデル動物の作出と治療法の開発 (2017)
  • Author(s): 今野 歩, 渡邊 将, 星野 千秋, 篠原 洋一郎, 松﨑 泰教, 平井 宏和
  • Organizer: 2017年度生命科学系学会合同年次大会（ConBio2017）
• [Presentation] AAV9 vector-mediated production of mouse model of spinocerebellar ataxia type 3 and electrophysiological analysis of that mouse (2017)
  • Author(s): Chiaki Hoshino, Masashi Watanabe, Ayumu Konno, Hirokazu Hirai
  • Organizer: 第40回日本神経科学大会
• [Presentation] Low concentration of a GABAb receptor agonist improves motor performance in spinocerebellar ataxia type 1 (SCA1) model mice (2017)
  • Author(s): Nobutake Hosoi, Shuvaev N Anton, Hirokazu Hirai
  • Organizer: 第40回日本神経科学大会
• [Presentation] Pros and cons of an AAV double infection method combined with the Tet system :An experimental study (2017)
  • Author(s): Nobutake Hosoi, Masayuki Shichida, Ayumu Konno, Hirokazu Hirai
  • Organizer: Neuroscience 2017
  • Int'l Joint Research
• [Presentation] Impaired synaptic plasticity at cerebellar parallel fiber-Purkinje cell synapses in AAV vector-based mouse model of spinocerebellar ataxia type 3 (2016)
  • Author(s): Masashi Watanabe, Ayumu Konno, Hirokazu Hirai
  • Organizer: 第３９回日本神経科学大会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-07-20
• [Remarks] 群馬大学大学院医学系研究科脳神経再生医学分野ホームページ
  • URL: http://synapse.dept.med.gunma-u.ac.jp/
• [Remarks] 赤色 cAMP 可視化蛍光タンパク質センサーの開発
  • URL: http://www.c.u-tokyo.ac.jp/info/news/topics/files/20170810pressrelease.pdf