These studies are designed to investigate the secretory potential of the keratinocyte and the possibility of genetically altering keratinocytes to deliver engineered proteins to the circulatory system. Sustained and efficient delivery of factors to the bloodstream by keratinocytes expressing a transgene has not yet been accomplished. Several current pitfalls will be addressed by: identifying promoters that are optimal for expressing transgenes in keratinocytes; exploring methods to obtain sustained high levels of transgene expression; and understanding secretion and diffusion of proteins through the dermo-epidermal junction. Three mammalian promoters, derived from keratin 1, keratin 6, and loricrin genomic DNA offer potential advantages since athe cellular machinery is already set up to express downstream sequences at high levels and the downregulation of viral promoters is avoided. These promoters will initially be studied in transgenic mice. Transgenic skin is intact and expected to form normally. Thus immunocytochemical and histological studies can be used to identify the specific cells that secrete protein, the pathways for secretion, and any buildup of secretion products. In addition with germline gene transfer, serum levels are expected well into the ELISA range enabling determination of the efficacy of the three promoters. The promoters that drive high expression in transgenic mice will then be incorporated into the design of expression vectors derived from bovine papillomaviruses (BPV). These viruses are maintained in host cells as multicopy episomes and thus avoid athe hazards of integration into host DNA. In addition, since 50 to 150 copies are potentially housed in each transfected cell, expression levels are expected to be high. The vectors will be stably transfected into mouse primary keratinocytes and sheets of transformed keratinocytes grafted onto mice. Serum levels of the engineered protein will then be assayed. In the final phases of this work, two in vivo delivery methods (hypospray and ballistic transfer) will be explored. In these studies, transgene DNA will be delivered directly tot he living animal, thus bypassing the requirement for primary culture and skin grafting. The candidate is a faculty member in the Department of Pediatrics, who hopes to develop into an independent investigator in the area of gene therapy. With the support of the Pediatrics, who hopes to develop into an independent investigator in the area of gene therapy. With the support of the Pediatric Department, she has completed didactic graduate level courses in molecular biology. Currently she is beginning a period of supervised research as outlined in this proposal to obtain intensive laboratory experience in the area of gene therapy. The work described here will be completed with the guidance of Dennis Roop, Ph.D., Professor of Cell Biology of Dermatology, who has trained several M.D.s and Ph.D.s who now have independent investigative careers.
