9612334 Lauffenburger In spite of their efficient gene transfer capabilities, current viral vectors fall short of ideal vector for human in-vivo gene therapy. Among the most serious problems are the random gene insertion into the host chromosome, immune responses caused by the natural viral proteins, and low cell selectivity and efficiency in the transfer. Also, neither adenoviral nor retrovial vectors can incorporate genes over 7 or 8 kb in size. There is a growing body of work demonstrating that certain ligands have the ability to bind DNA and effect its transfer into cells through very selective surface receptor binding. Thus conjugate gene delivery offers and improved delivery potential but has not been studied in enough detail to demonstrate feasibility. For gene transfer use. This proposal seeks to gain information on the rate- limiting steps of the receptor-mediated DNA/ligand- conjugate transport from the cell surface to the nucleus. Specific aims include: A. Determine whether cellular internalization is a limiting factor; B. Determine where in the endosomal sorting pathways conjugates escape to the cytosol; C. Predict and test desirable ligand properties to effect the transfer. The program will study the transfer of the epidermal growth factor (EGF) gene into mouse fibroblast cells that contain previously transfected EGF receptors. This robust system will allow variation of all the key factors. The P.I.'s lab has extensive experience working with the EGF system and has already demonstrated reasonable success transferring EGF to the mouse fibroblasts. ***
