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Development of novel recombinant enzyme for understanding complex brain development

Research Project

Project/Area Number 20K06464
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 42040:Laboratory animal science-related
Research InstitutionThe University of Tokyo

Principal Investigator

Tsunekawa Yuji  東京大学, 医科学研究所, 助教 (80733352)

Project Period (FY) 2020-04-01 – 2024-03-31
Project Status Completed (Fiscal Year 2023)
Budget Amount *help
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2022: ¥260,000 (Direct Cost: ¥200,000、Indirect Cost: ¥60,000)
Fiscal Year 2021: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2020: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
KeywordsAAV / LsCre / Creノックイン / Cre de novo ノックイン / ノックイン / リコンビネース
Outline of Research at the Start

ヒト型のシワのある大脳皮質がどのように発生するかを明らかにするにはマウス以外の高等哺乳類モデル動物を使う必要がある。しかし、それらの動物ではコンディショナルノックアウト (cKO)、リニエージトレーシング、機能獲得実験などの基本技術に必須であるリコンビネースをノックインすることができない。本研究では申請者の開発した体細胞に対するゲノム編集技術を改良し、食肉目のフェレットを複雑脳モデルとして、発生期においてcKO、リニエージトレーシング、細胞特異的機能獲得実験を可能にするCre-LoxPシステムの適用を目指す。

Outline of Final Research Achievements

We aimed to develop a technology to achieve cell-specific gene expression in various animal species to accommodate the difficulty in creating knock-in (KI) model animals for genetically engineering cell-specific gene expression. During knock-in, a phenomenon was observed in which Cre introduced into cells as a donor was unintentionally expressed, resulting in gene expression in cells other than the target cells. Therefore, we developed leakless Cre with codon modification to suppress nonspecific gene expression from the donor until knock-in is performed. We also developed a technology to efficiently produce and purify AAV by incorporating LsCre into adeno-associated virus (AAV), which is capable of systemic gene transfer, and reported the results.

Academic Significance and Societal Importance of the Research Achievements

研究の過程で開発し、論文として報告したアデノ随伴ウイルス(AAV)ベクターの新規高効率精製法は、濃縮、バッファー置換に用いる中空糸を用いた全く新規の精製方法であり、今後AAVベクターを用いた研究、遺伝子治療を加速させる学術的、社会的意義の高い成果だと考えられる。

Report

(5 results)
  • 2023 Annual Research Report   Final Research Report ( PDF )
  • 2022 Research-status Report
  • 2021 Research-status Report
  • 2020 Research-status Report
  • Research Products

    (6 results)

All 2023 2022

All Journal Article (5 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 5 results,  Open Access: 4 results) Patent(Industrial Property Rights) (1 results)

  • [Journal Article] Large-scale purification of functional AAV particles packaging the full genome using short-term ultracentrifugation with a zonal rotor2023

    • Author(s)
      Wada M, Uchida N, Posadas-Herrera G, Hayashita-Kinoh H, Tsunekawa Y, Hirai Y, Okada T.
    • Journal Title

      Gene Therapy

      Volume: Mar 28 Issue: 7-8 Pages: 641-648

    • DOI

      10.1038/s41434-023-00398-x

    • Related Report
      2023 Annual Research Report 2022 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Development of a novel purification method for AAV vectors using tangential flow filtration2023

    • Author(s)
      Miyaoka Rimi、Tsunekawa Yuji、Kurosawa Yae、Sasaki Takako、Onodera Azusa、Sakamoto Kenji、Kakiuchi Yuko、Wada Mikako、Nitahara‐Kasahara Yuko、Hayashita‐Kinoh Hiromi、Okada Takashi
    • Journal Title

      Biotechnology and Bioengineering

      Volume: 120 Issue: 11 Pages: 3311-3321

    • DOI

      10.1002/bit.28524

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Engineering a highly durable adeno-associated virus receptor for analytical applications2023

    • Author(s)
      Yoshida Kouhei、Tsunekawa Yuji、Kurihara Kento、Watanabe Kazuya、Makino-Manabe Yuriko、Wada Mikako、Tanaka Toru、Ide Teruhiko、Okada Takashi
    • Journal Title

      Molecular Therapy - Methods & Clinical Development

      Volume: 31 Pages: 101157-101157

    • DOI

      10.1016/j.omtm.2023.101157

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits2023

    • Author(s)
      Li Dan、Johmura Yoshikazu、Morimoto Satoru et al.
    • Journal Title

      Nature Aging

      Volume: 3 Issue: 8 Pages: 1001-1019

    • DOI

      10.1038/s43587-023-00464-4

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Subcellular mRNA localization and local translation of Arhgap11a in radial glial progenitors regulates cortical development2023

    • Author(s)
      Pilaz LJ, Liu J, Joshi K, Tsunekawa Y, Musso CM, D'Arcy BR, Suzuki IK, Alsina FC, Kc P, Sethi S, Vanderhaeghen P, Polleux F, Silver DL.
    • Journal Title

      Neuron .

      Volume: 111(6) Issue: 6 Pages: 839-856

    • DOI

      10.1016/j.neuron.2023.02.023

    • Related Report
      2022 Research-status Report
    • Peer Reviewed / Int'l Joint Research
  • [Patent(Industrial Property Rights)] ウイルスの精製方法2022

    • Inventor(s)
      恒川雄二
    • Industrial Property Rights Holder
      恒川雄二
    • Industrial Property Rights Type
      特許
    • Industrial Property Number
      2022-047745
    • Filing Date
      2022
    • Related Report
      2021 Research-status Report

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Published: 2020-04-28   Modified: 2025-01-30  

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