LATmiRNA

• Project/Area Number: 16K15093
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Applied molecular and cellular biology
• Research Institution: Nippon Medical School
• Principal Investigator: Okada Takashi 日本医科大学, 大学院医学研究科, 大学院教授 (00326828)
• Co-Investigator(Kenkyū-buntansha): 宮川 世志幸 日本医科大学, 医学部, 講師 (90415604)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: HSVベクター / CRISPR-Cas / SCA6 / 脊髄小脳失調症 / 遺伝子治療 / CRISPRi / CACNA1A / ヘルペスウイルス / 脊髄小脳変性症 / CRISPR-dCas9 / 脊髄小脳変性症６型 / CRISPR-Cas9 / 遺伝子編集 / LAT / TR

Outline of Final Research Achievements

In this study, we developed a method for gene knockdown of CACNA1A which is the responsible gene for spinocerebellar ataxia type 6 (SCA6) using CRISPR interference（CRISPRi）technology. We also engineered non-toxic herpes simplex virus-based (HSV) vectors encoding the CACNA1A-CRISPRi expression system. Our results would help develop for non-toxic HSV vector-mediated SCA6 therapy.

Academic Significance and Societal Importance of the Research Achievements

本研究の特色は、これまでに医療応用が難しかったHSVベクターを無毒化することで、CRISPR技術との融合を可能にした点である。HSVベクターは遺伝子治療ベクターとして研究開発されるようになり久しいが、それにも拘わらずその応用範囲が極めて限定的であったのは、その高い細胞障害性が原因であった。申請者らが開発した無毒化HSVベクターは、細胞障害性を完全に排除しているため、安全かつ効率的にCRISPR-Cas9システムを神経細胞に導入することができる。本技術が確立すれば、これまで根治が不可能であった脊髄小脳失調症6型（SCA6）などの神経変性疾患に対する治療技術の有力な選択肢の一つとなり得る。

Research Products

• [Journal Article] Supplemental Treatment for Huntington’s Disease with miR-132 that Is Deficient in Huntington’s Disease Brain (2018)
  • Author(s): Fukuoka Masashi、Takahashi Masaki、Fujita Hiromi、Chiyo Tomoko、Popiel H. Akiko、Watanabe Shoko、Furuya Hirokazu、Murata Miho、Wada Keiji、Okada Takashi、Nagai Yoshitaka、Hohjoh Hirohiko
  • Journal Title: Mol. Ther. Nucleic Acids Volume: 11 Pages: 79-90
  • DOI: 10.1016/j.omtn.2018.01.007
  • Peer Reviewed / Open Access
• [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] Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System (2018)
  • Author(s): Artusi Sara、Miyagawa Yoshitaka、Goins William、Cohen Justus、Glorioso Joseph
  • Journal Title: Diseases Volume: 6 Issue: 3 Pages: 74-74
  • DOI: 10.3390/diseases6030074
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Engineered HSV vector achieves safe long-term transgene expression in the central nervous system (2017)
  • Author(s): Verlengia Gianluca、Miyagawa Yoshitaka、Ingusci Selene、Cohen Justus B.、Simonato Michele、Glorioso Joseph C.
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 15071-11
  • DOI: 10.1038/s41598-017-01635-1
• [Presentation] AAVベクターを用いた遺伝子治療とゲノム医療 (2018)
  • Author(s): Okada T.
  • Organizer: 第50回日本臨床分子形態学会総会 Symposium 「ゲノムで見える病態」
  • Invited
• [Presentation] Enhancement of non-toxic HSV vector production and function by chemical compound treatment. (2018)
  • Author(s): Kuroda S, Miyagawa Y, Adachi K, Yamamoto M, Hayashita-Kinoh H, Cohen JB, Glorioso JC, Suzuki H, Yoshida H, Okada T.
  • Organizer: 第24回日本遺伝子細胞治療学会学術集会
• [Presentation] In vivo distribution of transgene expression from an HSV vector functionally deleted for all immediate-early genes (2018)
  • Author(s): Miyagawa Y, Maruyama M, Kuroda S, Sakai A, Sato Y, Hayashita-kinoh H, Yamamoto M, Cohen JB, Glorioso JC, Okada T.
  • Organizer: The 41th Annual meeting of the Molecular Biology Society of Japan
• [Presentation] 難治性神経・筋疾患の遺伝子治療に向けた新規無毒化ヘルペスウイルスベクターの開発. (2017)
  • Author(s): 宮川 世志幸、黒田 誠司、丸山 基世、喜納 裕美、山本 基子、Gianluca Verlengia、Michele Simonato、Justus Cohen、Joseph Glorioso、岡田 尚巳
  • Organizer: 2017年度生命科学系学会合同年次大会(ConBio2017)
  • Invited
• [Presentation] Persistent transduction of CNS neurons by a non-toxic herpes simplex virus-based vector. (2017)
  • Author(s): Yoshitaka Miyagawa, Gianluca Verlengia, Michele Simonato, Justus B. Cohen and Joseph C. Glorioso.
  • Organizer: The 23rd Annual Meeting of Japan Society of Gene and Cell Therapy
• [Presentation] Effect of culture conditions on efficient production of non-toxic herpes simplex virus-based vectors using a novel producer cell line. (2017)
  • Author(s): Seiji Kuroda, Yoshitaka Miyagawa, Kumi Adachi, Motoko Yamaoto, Justus B. Cohen, Joseph C. Glorioso, Hideyuki Suzuki, Eiji Uchida and Takashi Okada.
  • Organizer: The 23rd Annual Meeting of Japan Society of Gene and Cell Therapy
• [Presentation] Protocol optimization for high-yield production of a non-toxic herpes simplex virus-based vector using a novel producer cell line (2016)
  • Author(s): Kuroda S, Miyagawa Y, Adachi K, Yamamoto M, Cohen JB, Glorioso JC, Suzuki H, Uchida E and Okada T
  • Organizer: 第３９回日本分子生物学会
  • Place of Presentation: 横浜
  • Year and Date: 2016-11-30
• [Presentation] Deletion of virion host shut off protein promotes neural-specific transgene expression by a non-cytotoxic herpes simplex virus-based vector (2016)
  • Author(s): Miyagawa Y, Verlengia G, Simonato M, Cohen JB, Glorioso JC
  • Organizer: 第３９回日本分子生物学会
  • Place of Presentation: 横浜
  • Year and Date: 2016-11-30