期刊
MOLECULAR THERAPY-NUCLEIC ACIDS
卷 12, 期 -, 页码 554-567出版社
CELL PRESS
DOI: 10.1016/j.omtn.2018.06.008
关键词
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资金
- Institut National de la Sante et de la Recherche Medicale (INSERM)
- Dystrophic Epidermolysis Bullosa Association (DEBRA) France
- Association Francaise contre les Myopathies (AFM)
- European Research Council (ERC-2010-AdG, GT-SKIN)
- AFM (DEBTALENS-AFM)
Recessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.
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