An ex vivo keratinocyte model for gene therapy of hemophilia B.
We are investigating whether skin-targeted gene therapy may be used to treat hemophilia B by transplanting keratinocytes transduced by factor IX-expressing retroviral vectors. No pre-clinical animal model for keratinocyte-mediated gene therapy has shown long-term efficacy in vivo. It remains unclear whether this short-term expression is due to promoter shut-off or a reduced survival of grafted genetically modified cells. The purpose of this study was to determine the fate of primary human keratinocytes superficially grafted to nude mice in a silicone transplantation chamber. In addition, vectors containing keratinocyte-specific enhancers from the human papilloma virus-16 (HPV-16) and human keratin 5 and 14 genes were used upstream of the cytomegaloviral (CMV) immediate-early promoter/enhancer to control factor IX cDNA expression to avoid promoter shut-off. Factor IX was secreted by cultured keratinocytes after transduction by each of these chimeric promoter/enhancer vectors, although the levels varied according to the particular construct used. Keratinocytes transduced by the vector containing the HPV-16 enhancer were grafted into nude mice, and human factor IX was detected in plasma at 0.02-9 ng per ml for 4-5 wk for the duration of graft survival. The HPV-16 enhancer may be a useful addition to expression vectors for keratinocyte gene therapy. The transplantation chamber can be adapted to grafting retrovirally transduced keratinocytes for gene transfer studies.
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Sir William Dunn School of Pathology, University of Oxford, U.K.
Brownlee
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D000818:Animals; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism; D004195:Disease Models, Animal; D005164:Factor IX / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015316:Genetic Therapy; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D015603:Keratinocytes / Q000637:transplantation*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
D017077:Culture Media, Conditioned; D005164:Factor IX