Gene editing for corneal dystrophy using CRISPR/Cas9

• Project/Area Number: 17K11446
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Ophthalmology
• Research Institution: The University of Tokyo
• Principal Investigator: Usui Tomohiko 東京大学, 医学部附属病院, 届出診療員 (80282557)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 角膜ジストロフィ / 遺伝子編集 / 角膜ジストロフィー / 角膜 / 遺伝子 / 角膜混濁

Outline of Final Research Achievements

Granular corneal dystrophy (GCD) is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene. To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI. An R124H mutation in human corneal keratocytes derived from a GCD patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. No off-target effects were detected.
R124C mutation in TGFBI causes lattice corneal dystrophy type 1. We used CRISPR-Cas9 to establish the Tgfbi mutant mouse model. TGFBI-R124C mice were generated using ssODN-mediated base-pair substitution introduced via CRISPR-Cas9. Mice carrying base substitution in Tgfbi showed high frequency of corneal opacity. Corneal epithelial wound healing was affected by TGFBI-R124C mutation. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.

Academic Significance and Societal Importance of the Research Achievements

本課題における検討で、CRISPR-Cas9システムは、TGFBI遺伝子の点変異をin vitroにおいて正常に編集可能なことを示した。現在外科的治療では再発が必発であり、薬物治療法が全く存在しないTGFBI角膜ジストロフィの根治療法開発の基盤となる。
また高高率に角膜混濁が生じる本疾患のモデル動物は存在しなかったが、我々が作製したマウスは高率に角膜混濁を生じ、今後、遺伝子治療含め、本疾患に対する治療効果の検討をin vivoで行う意味で大変有用なモデル動物である。また現在までよく分かっていないTGFBI角膜ジストロフィにおける角膜混濁のメカニズム解明にも有用であると考えられる。

Research Products

• [Int'l Joint Research] Salk Institute for Biological Studies(米国)
• [Journal Article] Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair (2020)
  • Author(s): Kitamoto Kohdai、Taketani Yukako、Fujii Wataru、Inamochi Aya、Toyono Tetsuya、Miyai Takashi、Yamagami Satoru、Kuroda Masahiko、Usui Tomohiko、Ouchi Yasuo
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 2000-2000
  • DOI: 10.1038/s41598-020-58876-w
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] CRISPR・Cas9を用いた角膜変性症治療の可能性 (2018)
  • Author(s): 臼井 智彦
  • Journal Title: 日本の眼科 Volume: 89 Pages: 1254-1255
• [Journal Article] Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair (2017)
  • Author(s): Taketani Yukako、Kitamoto Kohdai、Sakisaka Toshihiro、Kimakura Mikiko、Toyono Tetsuya、Yamagami Satoru、Amano Shiro、Kuroda Masahiko、Moore Tara、Usui Tomohiko、Ouchi Yasuo
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 16713-16713
  • DOI: 10.1038/s41598-017-16308-2
  • Peer Reviewed / Open Access / Int'l Joint Research