Therapeutic Application of CRISPR-Cas3 for dominant dystrophic Epidermolysis Bullosa

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K16230
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 53050:Dermatology-related
• Research Institution: Osaka University (2022-2023); Kochi University (2021)
• Principal Investigator: Morisaka Hiroyuki 大阪大学, 大学院医学系研究科, 特任助教(常勤) (60826840)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: CRISPR / 表皮水疱症 / Gene editing / Cas3

Outline of Final Research Achievements

Epidermolysis bullosa is a serious disease characterized by genetic abnormalities, however, there is still no curative treatment. Recently, we have developed a gene editing tool called CRISPR-Cas3 that causes large deletions in the genome. We hypothesized it would be relevant to therapeutic benefit for dominant dystrophic epidermolysis bullosa (DDEB), which is a dominant-negative disease, if the mutated allele is successfully skipped by Cas3 gene editing. To establish a disease model, Cas9 protein were transfected to immortalized human keratinocyte cells. Sanger sequencing after cloning revealed clone 1 had 6nt deletion including a glycine codon in one allele, and clone 2 had premature stop codons in both alleles. Immunofluorescence staining and western blot showed almost no type VII collagen in clone2, whereas Clone1 had type VII collagen. These results suggest that clone 1 and clone 2 may be cell models for DDEB and recessive dystrophic epidermolysis bullosa, respectively.

Free Research Field

皮膚科学

Academic Significance and Societal Importance of the Research Achievements

遺伝子治療は以前から待望されていたが、CRIPSRの登場により現実的な治療となってきている。しかし、Cas9は米国が特許を持ち、日本の研究開発は後れを取っている現状がある。表皮水疱症を含むドミナントネガティブな疾患に対する変異アレルの欠失は、Cas9を含め既存の技術では困難であり、国産技術であるCRISPR-Cas3により始めて実現可能になる。本研究成果により、CRISPR-Cas3の遺伝子治療を評価する細胞モデルが確立できた。また、本研究により、表皮水疱症以外のドミナントネガティブな疾患への応用も十分に考えられ、今後の実際の医療への展開も大きく期待できる。