? The current project seeks to understand retrovirus-mediated gene transfer and to enhace the transduction efficiency to epidermal stem cells. Although retroviral transduction results in permanent genetic modification, differentiation and eventually loss of the transduced cells from the epidermis results in temporary transgene expression. Therefore, to achieve stable long-term gene expression, it is critical that the epidermal stem cells are transduced with high efficiency. Recent results from our laboratory show that gene transfer on fibronectin is significantly higher than on tissue culture plastic and implicate integrins in retroviral transduction of epidermal keratinocytes. Specifically, the efficiency of gene transfer correlates with the levels of alpha 5 and beta 1 integrins on the cell surface and blocking these integrins with antibodies decreases gene transfer significantly. We hypothesize that integrins may enhance gene transfer by enhancing the rate of keratinocyte migration on fibronectin and therefore the probability that itarget cells encounter the immobilized virus. We propose to develop two assays to test this hypothesis and use flow cytometry to identify other integrins that may also play a role in retroviral gene transfer. Since integr ns and attachment to extracellular matrix have been shown to characterize keratinocyte stem cell phenotype, we also propose to test the hypothesis that retroviral transduction on fibronectin increases the efficiency of gene transfer to epidermal stem cells. To this end we will use biochemical (e.g. flow cytometry) and functional assays (e.g. clonal analysis), as well as in vivo transplantation of genetically modified skin equivalents onto athymic mice. Finally we propose to engineer micropatterned surfaces and microfluidic networks to enhance deposition of retrovirus to the surface. Mathematical modeling and experiments will be employed to design the gene transfer """"""""chips"""""""" in order to increase the efficiency of gene transfer and control the number of gene copies per target cell. Reaching these goals will have significant impact on the potential of genetically modified cells to treat short or long-term disease states. ? ?
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