The anatomic location of the endothelial cell makes it an ideal target for in vivo gene transfer with consequent genetic modification of the endothelial cell for a variety of therapeutic purposes, including induction of angiogenesis, prevention of restenosis, following angioplasty, suppression of vessel growth in tumors, and as a source of therapeutic proteins for treatment of hereditary and acquired disorders. Of all gene transfer vectors available, adenovirus has been most effective at mediating endothelial gene transfer, but high vector concentrations are necessary, with the attendant risks for adverse events related to the vector. In this context, strategies using adenovirus vectors to transfer genes to endothelial cells could benefit from improved efficiency of gene transfer to endothelial cells which would lead to a corresponding decrease in dose of vector delivered to the patient. To realize the potential for increasing gene transfer to endothelial cells, the biology of gene transfer to endothelial cells must first by characterized. In this context, the central hypothesis of this proposal is that the evaluation of adenovirus trafficking in endothelial cells will yield strategies for improved efficiency of gene transfer to endothelial cells. To pursue this hypothesis, adenovirus interaction with endothelial cells will be evaluated in the context of varying physiological states of endothelial cells including resting or activated endothelial cells, as sub-confluent cultures, confluent polarized monolayers, or wounded polarized monolayers. These strategies will lay a foundation for understanding the impact of altered adenovirus tropism on efficiency of gene transfer to endothelial cells. This project is focused on two aims: (1) To characterize efficiency of adenovirus binding and intracellular trafficking in varying physiological states of endothelial cells; and (2) To characterize adenovirus entry and trafficking in endothelial cells subsequent to infection via alternate cell surface receptors.
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