Highly Efficient Ex Vivo Correction of COL7A1 through Ribonucleoprotein-Based CRISPR/Cas9 and Homology-Directed...

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of variants in COL7A1 encoding type VII Collagen, the major component of anchoring fibrils that form key attachment structures for dermal-epidermal adherence. In this study, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes and fibroblasts from 2 patients homozygous for the c.6508C>T (p.Gln2170∗) variant through CRISPR/Cas9-mediated homology-directed repair. Three guide RNAs targeting the c.6508C>T variant or harboring sequences were delivered together with high-fidelity Cas9 as a ribonucleoprotein complex. Among them, one achieved 73% cleavage activity in primary RDEB keratinocytes and RDEB fibroblasts. Then, we treated RDEB keratinocytes and RDEB fibroblasts with this specific ribonucleoprotein complex and the corresponding donor template delivered as single-stranded oligodeoxynucleotide and achieved up to 58% of genetic correction as well as type VII Collagen rescue. Finally, grafting of corrected 3-dimensional skin onto nude mice induced re-expression and normal localization of type VII Collagen as well as anchoring fibril formation at the dermal-epidermal junction 5 and 10 weeks after grafting. With this promising nonviral approach, we achieved therapeutically relevant specific gene editing that could be applicable to all variants in exon 80 of COL7A1 in primary RDEB cells.