Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a skin fragility disorder associated with mutations in COL7A1 gene. A highly prevalent mutation in Spanish RDEB patients’ cohort, c.6527insC, is located in exon 80 and results in a frame shift causing a premature termination codon. Homozygotic carriers of this mutation have no Collagen VII (C7) protein at the dermal-epidermal junction.
We had previously designed TALE Nucleases (TALENs) expressed by adenoviral vectors (Ad-TALENs) to generate indels in the vicinity of the c.6527insC mutation with the potential to restore COL7A1 gene reading frame and Collagen VII protein expression  as determined in an immortalized keratinocyte cell line (Chamorro et al., Mol Ther Nucleic Acids. 2016, Apr 5;5:e307). After infecting primary patient keratinocyte cultures with these Ad-TALENs, clones (n=136) with epidermal stem cell features were isolated based on their proliferation ability and analyzed phenotypically for restored C7 expression. All C7 positive clones (n=13) were shown to carry different reading frame-correcting indels. Selected clones were expanded and cells used to produce skin equivalents that were grafted onto immunodeficient mice to generate gene edited human skin. A single base pair deletion at the dsDNA break site generated by our TALENs was found to be the most common indel in genotyped clones. This deletion causes frame recovery but results in four amino acidic changes in the C7 protein that disrupt the Gly-X-Y pattern, characteristic of the triple helix-forming domain, and hinders protein secretion. Skin equivalents generated with these clones displayed C7 at the basal layer of epidermis, but not at the basement membrane zone, and exhibited blisters at the dermal-epidermal junction. A clone carrying a bigger deletion encompassing exon 80 entirely was found to express a truncated C7 lacking exon 80 encoded sequence. Skin equivalents generated with this clone regenerated human skin displaying ColVII at the basement membrane zone, dermal-epidermal adhesion as a consequence of anchoring fibrils formation. Our results indicate that it is possible to edit the COL7A1 gen in primary patient stem cells, isolate edited clones and use them to generate functional human skin with therapeutic potential. We also demonstrate the structural functionality of C7 lacking exon 80 encoded sequence and therefore the utility of exon-deleting strategies to restore ColVII protein deposition at the dermal-epidermal junction and revert the RDEB phenotype.

Gene edition-mediated deletion of a mutation-containing COL7A1 exon 80 in epidermal stem cells of RDEB patients allows regeneration of normally adhesive skin