論文

査読有り 国際誌
2020年6月

Footprint-free gene mutation correction in induced pluripotent stem cell (iPSC) derived from recessive dystrophic epidermolysis bullosa (RDEB) using the CRISPR/Cas9 and piggyBac transposon system.

Journal of dermatological science
  • Munenari Itoh
  • ,
  • Shiho Kawagoe
  • ,
  • Katsuto Tamai
  • ,
  • Hidemi Nakagawa
  • ,
  • Akihiko Asahina
  • ,
  • Hirotaka James Okano

98
3
開始ページ
163
終了ページ
172
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1016/j.jdermsci.2020.04.004

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic skin blistering disorder caused by mutations in the type VII collagen gene. A combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to develop gene and cell therapies for such inherited diseases. However, the methodologies for gene and cell therapies must be continuously innovated for safe clinical use. OBJECTIVE: In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and the piggyBac transposon system so that no residual gene fragments remained in the genome of iPSCs after correcting the mutation. METHODS: For homologous recombination (HR)-based gene editing using CRISPR/Cas9, we designed guide RNA and template DNA including homologous sequences with drug-mediated selection cassette flanked by inverted repeat sequences of the transposon. HR reaction using CRISPR/Cas9 was induced in RDEB-specific iPSCs, and mutation-corrected iPSCs (MC-iPSCs) was obtained. Consequently, the selection cassette in the genome of MC-iPSCs was removed by transposase expression. RESULTS: After CRISPR/Cas9-induced gene editing, we confirmed that the pathogenic mutation in RDEB-specific iPSCs was properly corrected. In addition, MC-iPSCs had no genetic footprint after removing the selection cassette by transposon system, and maintained their "stemness". When differentiating MC-iPSCs into keratinocytes, the expression of type VII collagen was restored. CONCLUSIONS: Our study demonstrated one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for future clinical use.

リンク情報
DOI
https://doi.org/10.1016/j.jdermsci.2020.04.004
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/32376152

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