Construction of Skin Equivalents for Gene Therapy of Recessive Dystrophic Epidermolysis Bullosa

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Abstract
We have shown that retroviral vectors efficiently transfer the 9-kb collagen type VII cDNA into keratinocytes of dogs with recessive dystrophic epidermolysis bullosa (RDEB) and achieve correction of the RDEB phenotype in vitro. As a next step toward gene therapy applications, we have assessed the suitability of retroviral vectors to transduce human collagen type VII cDNA into primary human RDEB keratinocytes and generate transplantable autologous skin equivalents. The transduced RDEB keratinocytes permanently express high levels of recombinant collagen type VII that assembles into functional homotrimers readily secreted into the extracellular matrix. The recombinant collagen type VII reverts the migration and invasion potential of the transduced RDEB keratinocytes in vitro and is efficiently deposited at the dermal epidermal junction of artificial skin prepared with the reverted cells and artificial dermis made of biomaterial sponges embedded with dermal RDEB fibroblasts. Transplantable fibrin-based skin equivalents made with the transduced RDEB keratinocytes and grafted onto SCID mice either orthotopically or in accordance with the flap method generated cohesive and orderly stratified epithelia with all the characteristics of normal human epidermis, including rapid formation of anchoring fibrils. Because transplantable epithelia are routinely used to cure patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary RDEB epidermal clonogenic cells mediated by recombinant retroviral vectors opens new perspectives in the long-term treatment of genodermatoses.