Development of expression analysis for retinitis pigmentosa causative genes using peripheral blood by epigenome editing

• Project/Area Number: 20K09765
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 56060:Ophthalmology-related
• Research Institution: Nagoya University
• Principal Investigator: Nishiguchi Koji 名古屋大学, 医学系研究科, 教授 (30447825)
• Co-Investigator(Kenkyū-buntansha): 中澤 徹 東北大学, 医学系研究科, 教授 (30361075) ; 藤田 幸輔 名古屋大学, 医学系研究科, 助教 (80708115)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2022: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2021: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
• Keywords: 網膜色素変性 / ゲノム編集 / リンパ球

Outline of Research at the Start

網膜色素変性（RP）患者由来の不死化リンパ球を対象に、ゲノム編集技術を用いて特定のRP病因遺伝子のプロモーターを非メチル化することにより同遺伝子の強制発現細胞株の樹立を可能にする技術を開発する。この細胞株を用いて、本来リンパ球では発現しないRP病因遺伝子のmRNA発現解析を行い、同患者のゲノム情報と照合することにより、逆算的に病因変異が同定可能か検証する。このアプローチの有用性が明らかになった場合、比較的簡便かつ安価な遺伝子診断補助検査法の開発が可能になり、遺伝性疾患の診断率の向上に寄与すると期待される。

Outline of Final Research Achievements

The genetic basis of Japanese retinitis pigmentosa remains largely unknown. Therefore, using genome editing technology, we developed a technique for over expression of a specific retinitis pigmentosa-causing gene promoter by unmethylating it in lymphoblasts derived from patients with retinitis pigmentosa. First, the EYS gene, which has the highest frequency as a pathogenic gene in Japanese patients with retinitis pigmentosa, was targeted for development. We performed mRNA analysis of the EYS gene expressed by genome editing in a patient-derived lymphoblastoid cell lines, and confirmed that the disease-causing mutation could be identified by comparing it with the patient's genomic information. It is possible to develop a comparatively simple and inexpensive genetic test method.

Academic Significance and Societal Importance of the Research Achievements

本邦の失明原因の上位である網膜色素変性は難治性の遺伝性疾患であるが、大半の患者で病因変異が特定できていない。mRNA解析が有効な補助診断となりえるが、血液など容易に採取可能な患者由来サンプルで有用な結果を得るのは困難であった。この状況において、本研究では、患者リンパ球由来の網膜特異的遺伝子の強制発現細胞株を樹立し、遺伝子解析に対する有効性を示すことができた。この技術が発展すれば遺伝子診断の補助検査として広く普及する可能性がある。さらに、本技術は、網膜疾患以外の遺伝性疾患や非遺伝性疾患の病態解明や治療開発にも応用可能であり、汎用性と波及効果が極めて高く、その意義は大きい。

Research Products

• [Int'l Joint Research] バーゼル大学(スイス)
• [Int'l Joint Research] University of Basel(スイス)
• [Int'l Joint Research] バーゼル大学(スイス)
• [Journal Article] A hypomorphic variant in EYS detected by genome-wide association study contributes toward retinitis pigmentosa. (2021)
  • Author(s): Nishiguchi KM, Miya F, Mori Y, Fujita K, Akiyama M, Kamatani T, Koyanagi Y, Sato K, Takigawa T, Ueno S, Tsugita M, Kunikata H, Cisarova K, Nishino J, Murakami A, Abe T, Momozawa Y, Terasaki H, Wada Y, Sonoda KH, Rivolta C, Tsunoda T, Tsujikawa M, Ikeda Y, Nakazawa T.
  • Journal Title: Commun Biol Volume: 4 Issue: 1 Pages: 140-140
  • DOI: 10.1038/s42003-021-01662-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Development of Genome Editing Gene Therapy for Inherited Retinal Degeneration (2021)
  • Author(s): Koji M Nishiguchi
  • Organizer: 1st Asia Retina Congress
  • Int'l Joint Research / Invited
• [Presentation] Application of a Novel Genome Editing Approach for Treating a Common Mutation in a Large Gene Untreatable by Conventional Gene Supplementation (2021)
  • Author(s): Koji M Nishiguchi
  • Organizer: Sonoma Eye Virtual Conference
  • Int'l Joint Research / Invited