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 retroviral vectors containing the factor IX cDNA under the control of a cytomegaloviral (CMV) promoter/enhancer alone or as hybrids with either 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 hybrid constructs were disproportionately more active in primary keratinocytes than in the basal-like HaCaT cells. After skin grafting to athymic mice, transduced HaCaT cells differentiated to form a stratified epidermis that remained viable for at least 99 days in some mice. Factor IX in plasma of mice grafted with vectors containing the HPV-16 and hK5 elements was two- to three-fold higher than with vectors containing the CMV promoter alone. These results are consistent with the expected up-regulation in differentiated suprabasal cells by the HPV-16 and hK5 elements. Enhancers may be useful in specifically targeting the differentiated layer of the epidermis or achieving higher levels of gene expression after transplantation.
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Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
Brownlee
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D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D017077:Culture Media, Conditioned; D004742:Enhancer Elements, Genetic; D000078404:Epidermal Cells; D005164:Factor IX / Q000096:biosynthesis*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D051379:Mice; D008819:Mice, Nude; D027383:Papillomaviridae / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D015854:Up-Regulation
D017077:Culture Media, Conditioned; D005164:Factor IX