The broad objective of this proposal is to develop proof of principle and establish the parameters for the external regulation of transgenes of genetically modified cells of the skin. Transplantation to the skin of keratinocytes containing transgenes has resulted in local and systemic delivery of transgene products. This approach is limited by the lack of control over the amount and the timing of release of the transgene product. To address this problem, transgene expression will be placed under control of a promoter whose activity is tightly modulated by exogenously administered tetracycline. It is hypothesized that keratinocytes transfected with this system can be transplanted to the skin and their transgene activities controlled by externally applied tetracycline analogs. The ultimate goal of this proposal is to use this methodology to correct deficient gene expression in disease states. Accordingly, the specific aims are: 1) Establishment of an in vitro system for tetracycline regulated gene expression in immortalized human keratinocytes in order to establish that tetracycline can regulate gene expression in immortalized human keratinocytes in order to establish that tetracycline can regulate gene expression in human keratinocytes; 2) Development of tetracycline-regulated lacZ expression by immortalized human keratinocytes in vivo. These studies will define the [parameters necessary for topically and systematically administered tetracycline regulation of transgene expression in transplanted human keratinocytes in vivo; 3) Characterization of tetracycline-regulated expression of transgene products in transplanted immortalized human keratinocytes by regulating keratinocyte expression of transgenes having local (keratinocyte growth factor modulation of hair growth) or systematic (erythropoietin control of hematocrit) effects; and 4) Reconstitution of deficient gene expression in vivo by treating the anemia due to renal insufficiency (5/6 nephrectomy) with transplanted immortalized human keratinocytes containing the erythropoietin gene under tetracycline control. These experiments will demonstrate the feasibility of correcting and regulating local and/or system disease with genetically modified keratinocytes; experience gained can be applied to other cells and other organs.
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