DIFFERENTIATION-SPECIFIC ENHANCER ACTIVITY IN TRANSDUCED KERATINOCYTES - A MODEL FOR EPIDERMAL GENE-THERAPY

HaCaT cells, a spontaneously immortalised, nontumorigenic keratinocyte line, were used as a more amenable ,model than primary keratinocytes for ex vivo-mediated gene transfer These cells were transduced with re troviral vectors containing the factor IX cDNA under the control of a cytomegaloviral (CMV) promoter/enhancer alone or as hybrids with eithe r the human papilloma virus-16 (HPV-16), keratin 14 (hK14) or keratin 5 (hK5) regulatory elements. Unlike primary keratinocytes, HaCaT cells tolerated transduction and G418 selection well. The HPV-16 and hK5 hy brid constructs were disproportionately more active in primary keratin ocytes than in the basal-like HaCaT cells. After skin grafting to athy mic mice, transduced HaCaT cells differentiated to form a stratified e pidermis that remained viable for at least 99 days in some mice. Facto r IX in plasma of mice grafted with vectors containing the HPV-16 and hK5 elements was true-to three-fold higher than with vectors containin g the CMV promoter alone. These results are consistent with the expect ed up-regulation in differentiated suprabasal cells by the HPV-16 and hK5 elements. Enhancers may be useful in specifically targeting the di fferentiated layer of the epidermis or achieving higher levels of gene expression after transplantation.