Genetic modification of cultured skin substitutes by transduction of humankeratinocytes and fibroblasts with platelet-derived growth factor-A

Gene therapy promises the potential for improved treatment of cutaneous wou nds. This study evaluated whether genetically modified cultured skin substi tutes can act as vehicles for gene therapy in an athymic mouse model of wou nd healing. Human keratinocytes and fibroblasts were genetically engineered by retroviral transduction to overexpress human platelet-derived growth fa ctor-A chain. Three types of skin substitutes were prepared from collagen-g lycosaminoglycan substrates populated with fibroblasts and keratinocytes: H F-/HK-, containing both unmodified fibroblasts and keratinocytes: HF-/HK+, containing unmodified fibroblasts and modified keratinocytes: and HF+/HK-, containing modified fibroblasts and unmodified keratinocytes, Skin substitu tes were cultured for two weeks before grafting to full-thickness wounds on athymic mice. The modified skin substitutes secreted significantly elevate d levels of platelet-derived growth factor throughout the culture period. E xpression of retroviral platelet-derived growth factor-A mRNA was maintaine d after grafting to mice, and was detected in all HF-/HK+ grafts and one HF +/HK- graft at two weeks after surgery. Although no differences were seen b etween control and modified grafts, the results suggest that genetically mo dified cultured skin substitutes can be a feasible mechanism for cutaneous gene therapy. The cultured skin model used for these studies has advantages over other skin analogs containing only epidermal cells: because it contai ns both fibroblasts and keratinocytes, it therefore offers greater opportun ities for genetic modification and potential modulation of wound healing.