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Mol Ther Nucleic Acids. 2018 Sep 7;12:554-567. doi: 10.1016/j.omtn.2018.06.008. Epub 2018 Jun 26.

Ex Vivo COL7A1 Correction for Recessive Dystrophic Epidermolysis Bullosa Using CRISPR/Cas9 and Homology-Directed Repair.

Author information

1
Laboratory of Genetic Skin Diseases, INSERM UMR 1163, Imagine Institute, 24 bd du Montparnasse, Paris, France; University Paris Descartes-Sorbonne Cité, Paris, France.
2
INSERM UMR 951, Genethon, Evry, France.
3
Electronic Microscopy Facility, INSERM UMR 1016, Cochin Institute, Paris, France.
4
University Paris Descartes-Sorbonne Cité, Paris, France; Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; Imagine Institute, Paris, France.
5
Laboratory of Genetic Skin Diseases, INSERM UMR 1163, Imagine Institute, 24 bd du Montparnasse, Paris, France; University Paris Descartes-Sorbonne Cité, Paris, France; Department of Genetics, Necker Hospital for Sick Children, APHP, Paris, France. Electronic address: alain.hovnanian@inserm.fr.

Abstract

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.

KEYWORDS:

COL7A1; CRISPR/Cas9; droplet digital PCR; gene targeting; homology-directed repair; primary keratinocytes and fibroblasts; recessive dystrophic epidermolysis bullosa; skin grafts

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