A study to elucidate influence of triplet repeat expansion on pathology in Fuchs endothelial corneal dystrophy

2021 Fiscal Year Final Research Report

• Project/Area Number: 18K16947
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 56060:Ophthalmology-related
• Research Institution: The University of Tokyo
• Principal Investigator: MIYAI TAKASHI 東京大学, 医学部附属病院, 講師 (40599007)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Keywords: 角膜ジストロフィ / ゲノム編集 / 角膜内皮 / Fuchs角膜内皮ジストロフィ / TCF4

Outline of Final Research Achievements

In this research, STR assay and repeat primed PCR were performed on the DNA of 41 outpatients with Fuchs corneal endothelial dystrophy, and repeat elongation of TCF4 was observed in 8 patients (19.5%).
We succeeded in cleaving the repeat of the immortalized corneal endothelial cell HCEnC using CRISPR-Cas9, and succeeded in creating a knock-out cell line. We also succeeded in producing knock-in cell lines for HCEnC with 60-repeat and 80-repeat donors using the Homology direct repair method. RNA foci was observed in the 80 repeat knock-in cell line, and TCF4 gene expression by RT-PCR was increased.

Free Research Field

眼科学

Academic Significance and Societal Importance of the Research Achievements

本課題における検討において、世界で角膜移植の約４０％を占めるFuchs角膜内皮変性症について本邦の症例のDNA解析を行い、TCF4遺伝子のリピート伸長型が一定の比率を占めることを確認できた。また、CRISPR-Cas9を用いて角膜内皮細胞株のリピートのノックアウトおよびノックインに成功し、リピート切断治療の可能性および、リピート伸長によるRNA凝集という細胞内の病態を反映するモデルを作成できた。今後のFuchs角膜内皮変性症の病態解明および治療法の検討にあたり、大変有用なモデル細胞で学術的、社会的意義があると考えられる。