In vivo gene editing based on CRISPR/Cas9 adenoviral vector delivery in humanized mouse models of Recessive Dystrophic Epidermolysis Bullosa Bullosa skin

M García(1,5) J Martinez(2) R xu(1) J Bonafont(1) F Larcher(1,5) M Del Rio(1,5) R Hernandez(3,4) M Garin(2,5) A Mencia(2,5) R Murillas(2,5)

1:UC3M-CIBERER-CIEMAT; 2:CIEMAT-CIBERER; 3:Universidad de Navarra; 4:CIMA; 5:FJD

Recessive Dystrophic Epidermolysis Bullosa Bullosa (RDEB), a devastating skin fragility disease characterized by recurrent skin blistering, scar formation and high risk of developing squamous cell carcinoma, is caused by mutations in COL7A1, the gene encoding anchoring fibril-forming Collagen VII. Deletion of small, in-phase exons encoding repetitive collagenous domain results in truncated Collagen VII fit for fibril formation.  We have generated helper-dependent adenoviral vectors for CRISPR/Cas9 delivery to delete exon 80 of COL7A1, which contains a frameshift mutation that is highly prevalent in the Spanish population of RDEB patients. This NHEJ-based editing strategy proved to be highly effective for the restoration of Collagen VII expression when tested in keratinocyte and fibroblast cultures from patients. To model in vivo delivery of the viral vector into patient skin tissue, we used a humanized skin mouse model generated by grafting patient skin equivalents onto immunodeficient mice. Excisional wounds on regenerated patient skin grafts were filled with the adenoviral vectors embedded in a fibrin gel. We detected Collagen VII deposition in the basement membrane zone of the wounded areas treated with the vectors, suggesting that RDEB patient skin lesions can be directly treated by CRISPR/Cas9 delivery in vivo.