Dystrophic epidermolysis bullosa (DEB) is an incurable, inherited mechano-bullous disease of the skin characterized by skin fragility, blister formation, and chronic wounds. DEB is caused by defects in the human gene encoding type VII collagen. Type VII collagen is the main component of anchoring fibrils, structures within the basement membrane of skin which are attenuated, diminutive, or absent in DEB. These structures are thought to anchor the epidermis to the dermis, and their paucity in DEB results in the skin features. Gene therapy is considerated one strategy to correct this defect. The long-term objective of this proposal is to develop an ex vivo gene therapy strategy for DEB. We propose the targeted introduction of a transgene coding for type VII collagen into cultured keratinocytes and fibroblasts from DEB patients, cells that cannot endogenously make this protein. The """"""""gene corrected"""""""" cell cultures would then be transplanted onto the patients as an autograft. We have constructed both of full-length type VII collagen and a truncated type VII minicollagen plasmid expression vectors and purified large quantities of the recombinant proteins from stably transfected human cells. The minicollagen although truncated retains all the functional properties of a full-length alpha chain. Using a retroviral-mediated expression vector, we have induced RDEB keratinocytes to express the minicollagen which reversed the RDEB cellular phenotype to that of normal keratincytes. In this proposal, we will build upon this work and develop highly efficient viral vectors that will allow sustained and targeted type VII collagen transgene delivery into human keratinocytes and fibroblasts. We will generate new minigene and full length type VII collagen delivery vehicles using lentiviral vectors which can offer high efficiency, sustained transgene expression and the ability to transduce nondividing cells and can accommodate the 9 kb full-length type VII collagen Cdna. We will also use vectors with cellular promoters, such as the K14 promoter, to promote sustained expression and target the basal keratinocytes. Using our well- established assays, we will examine the phenotype and cellular functions of """"""""gene-corrected"""""""" RDEB cells. Lastly, we will advance from our monolayer keratinocyte and fibroblast cultures to a three dimensional, composite, skin- equivalent, organotypic culture model with diseased and """"""""gene corrected"""""""" RDEB cells. These cultures will also be grafted onto SCID mice and evaluated for anchoring fibril formation and sustained expression of the transgenes for type VII collagen.
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