Low expression of therapeutic genes products is a problem that has plagued all approaches in human gene therapy. Much of the effort to overcome this problem has focused on increasing transduction efficiency, promoter activity and altering gene structure to enhance expression. An alternative approach is to increase the net """"""""activity"""""""" of gene products by increasing their transport through the producing tissue, decreasing their clearance from the circulation, or enhancing their stability and targeting to the site of action. To accomplish this, an understanding of fundamental protein transport mechanisms through tissues is required. We have defined several key molecular parameters that theoretically control transport of proteins through tissues and have identified endosomal dissociation and ligand recycling as particularly important. To critically test their importance, epidermal growth factor (EGF) will be systematically modified by site-directed mutagenesis to increase its dissociation from receptors at endosomal pH values. Chimeras between EGF and recycling proteins, such as transferrin, will also be made. The relative trafficking of modified EGF in cultured cells will be determined and compared to their transport through artificial tissues consisting of cells embedded in different matrices. These data will be used to refine a quantitative model of tissue transport. In addition, transport of modified ligands from cell grown to either a nylon matrix or hollow fibers will be followed. The ligand release rates from these devices in vitro will be compared to circulating protein levels following their implantation in athymic mice. This should indicate how context affects transport or different gene products. Finally the studies will be extended to a second type of ligand, keratinocyte growth factor, to determine the extend to which our concepts can be applied to other bioactive molecules. This project should provide basic information on effective strategies to increase activities of therapeutic gene products.
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