Original Article

Journal of Investigative Dermatology (1997) 109, 139–145; doi:10.1111/1523-1747.ep12319194

An Ex Vivo Keratinocyte Model for Gene Therapy of Hemophilia B

Sean M Page1 and George G Brownlee1

1Sir William Dunn School of Pathology, University of Oxford, Oxford, U.K.

Received 20 January 1997; Revised 25 April 1997; Accepted 7 May 1997.

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Abstract

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-1 6 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.

Keywords:

epidermis, skin grafting., factor IX, retroviral vector

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