EX-VIVO AND IN-VIVO GENE-TRANSFER TO THE SKIN USING REPLICATION-DEFICIENT RECOMBINANT ADENOVIRUS VECTORS

The skin has the potential for a variety of gene therapy applications. In addition to local delivery, it is the largest organ of the body, a nd highly vascular, and thus is an ideal site for systemic delivery of gene products. To evaluate the potential for adenovirus-mediated skin gene transfer, the replication-deficient recombinant adenovirus vecto rs Ad.RSV beta gal (coding for Escherichia coli beta-galactosidase) an d Ad alpha 1AT (coding for human alpha 1-antitrypsin) were used in bot h ex vivo and in vivo approaches. Following in vitro infection with Ad .RSV beta gal, murine keratinocytes expressed beta-galactosidase. Para llel in vitro studies with Ad alpha 1AT documented de novo synthesis a nd secretion of human alpha 1AT as shown by [S-35]methionine labeling and immunoprecipitation. Quantification of human alpha 1AT in the cult ure supernatants demonstrated 0.1-0.3 mu g human alpha 1AT secreted/ml -24 h. Evaluation of the serum of mice receiving transplants (10(5) ce lls/ mouse) of Ad alpha 1AT-infected syngeneic keratinocytes demonstra ted human alpha 1AT for at least 14 d with maximum levels of 41 ng/ml. To demonstrate the feasibility of direct adenovirus-mediated in vivo transfer of genes to the skin, Ad.RSV beta gal or Ad alpha 1AT were ad ministered subcutaneously to mice. Histologic evaluation after 4 d dem onstrated expression of beta-galactosidase in various types of skin ce lls. Quantification of human alpha 1AT in serum of animals infected su bcutaneously with Ad alpha 1AT showed levels of 53 ng/ml at day 4, wit h human alpha 1AT detectable for at least 14 d. These observations sup port the feasibility of ex vivo and in vivo gene transfer to the skin mediated by replication-deficient adenovirus vectors.