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 mutations in COL7A1 encoding type VII collagen, the major component of anchoring fibrils critical attachment structures that adhere the epidermis to the dermis. Here, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes, fibroblasts, and 3D-skin grafts through CRISPR/Cas9-mediated Homology Directed Repair. First, we designed three guide RNAs (gRNAs) targeting the specific mutation or sequences in close distance to the c.6508C>T (p.Gln2170*) mutation to be corrected in exon 80. Chemically modified gRNAs were delivered together with high fidelity Cas9 as a ribonucleoprotein complex by nucleofection (RNP). Among the gRNAs tested, one intronic gRNA achieved up to 73% cleavage activity in RDEB keratinocytes and fibroblasts. We also evaluated their off-target activity in RDEB cells and found no evidence for non-specific cleavage activity at the in-silico predicted sites. We treated RDEB keratinocytes and fibroblasts with site-specific RNPs together with the corresponding Donor delivered as ssODN without antibiotic or fluorescent-based selection. Up to 58 % of genetic correction and C7 rescue were observed in bulk-nucleofected RDEB keratinocytes and fibroblasts as assessed by allele-specific TaqMan-ddPCR and Western blotting. Grafting of genetically corrected 3D skin equivalents onto nude mice induced re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction at five and ten weeks post-grafting. Our study demonstrates a highly efficient and precise genome editing applicable to all mutations in exon 80 of COL7A1 in primary RDEB keratinocytes and fibroblasts. This approach has a potential for future ex-vivo clinical applications.

ESDR156 - Targeted ex-vivo correction of COL7A1 through RNP-based CRISPR/Cas9 and Homology-Directed Repair to treat recessive dystrophic epidermolysis bullosa

50th Annual Meeting of the European Society for Dermatological Research. VIRTUAL MEETING