Selection of keratinocytes transduced with the multidrug resistance gene in an in vitro skin model presents a strategy for enhancing gene expression in vivo

In gene therapy studies, achieving prolonged, high-level gene expression in a significant percentage of cells has been difficult. One solution to enha nce expression would be to select for cells expressing both the desired gen e and a linked selectable marker gene in a bicistronic vector. As a potenti al target tissue, the skin is easily accessible for safe topical applicatio n of a selecting agent that could lead to significant gene expression in a high percentage of keratinocytes. To test the feasibility of such an approa ch, a skin raft culture model was developed. Human keratinocytes were trans duced with the multidrug resistance (MDR) gene, which confers resistance to a variety of cytostatic and antimitotic compounds, such as colchicine. Whi le growing on acellular dermis, transduced keratinocytes were treated with various doses of colchicine (10-50 ng/ml). Colchicine treatment increased t he percentage of keratinocytes expressing MDR to almost 100% in raft cultur es, Significantly, keratinocytes in colchicine-treated, MDR-transduced raft cultures were able to proliferate normally and form a stratified, differen tiated epidermis. This model suggests that topical selection for MDR-expres sing keratinocytes in vivo should be feasible without hampering the biologi c integrity of skin. Thus, topical selection leading to enhanced expression of a desired gene, linked to a resistance gene, holds future promise for s kin gene therapy.