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.