Development of the platform of gene therapy for inherited retinal diseases using gene-editing technologies

2022 Fiscal Year Final Research Report

• Project/Area Number: 20K09823
• 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: The University of Tokyo
• Principal Investigator: Iwagawa Toshiro 東京大学, 医学部附属病院, 特任助教 (30638648)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: エクソンスキッピング / 遺伝子治療 / ヒトiPS細胞 / RPE / ゲノム編集

Outline of Final Research Achievements

In order to develop a treatment for inherited retinal diseases, I evaluated the effects of exon-skipping using a CRISPR/Cas9 system on retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells (iPSCs) with a mutation in CHM or MYO7A. Under oxidative stress, RPE with a mutation in CHM exon 6 showed the decreased expression level of CHM protein and phagocytic activity. By targeting a splice donor cite of exon 6, exon 6-skipping was induced and the expression of truncated CHM protein was confirmed. CHM exon 6-skipping partially rescued the decreased phagocytic activity and affected the localization of RAB38 protein. Therefore, CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress. In the future, the evaluation of the exon-skipping targeting MYO7A in RPE and retinal cells is hoped.

Free Research Field

眼科学

Academic Significance and Societal Importance of the Research Achievements

視細胞や網膜色素上皮（RPE）が変性する遺伝性網膜変性症（IRD）に対する根本的な治療法の開発を目指し、本研究は筋ジストロフィーで有効性が見出されているエクソンスキッピングの評価を試みた。ゲノム編集技術であるCRISPR/Cas9システムによりCHMやMYO7Aの機能を喪失するヒトiPS細胞変異株を樹立し、RPEへ分化させた後に、エクソンスキッピングを誘導した。CHMの変異によりRPEの機能低下が認められたが、エクソンスキッピングにより一部機能の回復が認められるとともに、RAB38の局在の変化が見出され、エクソンスキッピングの有効性が示唆された。研究成果は査読付き論文として発表した。