833 Ex vivo gene therapy of recessive dystrophic epidermolysis bullosa skin cells leads to dermo-epidermal adhesion strength restoration in autologous bilayered self-assembled skin substitutes

Although gene therapy shows great promises for the treatment of Recessive Dystrophic Epidermolysis Bullosa (RDEB), a rare genodermatosis (3/million births) in which minor mechanical stress to the skin results in blisters and scarring leading to severe pain, permanent restoration of normal type VII collagen (C7) by genetically modified cells remains a challenge. Here, we investigate the potential of gene therapy combined with autologous bilayered skin substitutes. Viral transduction efficiency of C7 in RDEB fibroblasts by a gamma-retrovirus was improved 2.6 folds (p<.05) when using EF-c peptide compared to using polybrene and did not affect fibroblasts proliferation and keratinocytes stem cell content whereas polybrene led to a decrease in both (-20% and -33%, respectively (p<.01)). Overall, we restored C7 production in 55% of fibroblasts and 65% of keratinocytes. Skin substitutes produced with various combinations of untreated and corrected RDEB cells showed that before grafting corrected fibroblasts allows for a higher deposition of C7 compared to corrected keratinocytes. Quantitative adhesion strength of the dermo-epidermal junction (DEJ) showed comparable mechanical properties of skin substitutes produced with healthy cells (2.9mN/mm) and those with corrected RDEB cells (3.9mN/mm), but not those produced with untreated RDEB cells (1.5mN/mm). After grafting on athymic mice, C7 deposition at the DEJ and stem keratinocytes were maintained for at least 11 months. After grafting, the surface area of corrected skin substitutes was increased by 4 folds compared to RDEB skin substitutes. In conclusion, the self-assembly approach shows great potential as a long-term treatment of RDEB wounds.