1. ¹Centre National de la Recherche Scientifique, Genomes and Cancers Unit, Institut Gustave Roussy, Villejuif, France
2. ²Université Paris-Sud, Villejuif, France
3. ³Cutaneous Disease Modeling Unit and Regenerative Medicine Unit, Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, Madrid, Spain
4. ⁴Institut National de la Santé et de la Recherche Médicale U781, Bâtiment Maurice Lamy Hôpital Necker-Enfants Malades, Paris, France

Correspondence: Thierry Magnaldo, Institut Gustave Roussy, Centre National de la Recherche Scientifique, FRE 2939, Genomes and Cancers Unit, 39, rue Camille Desmoulins, Villejuif 94805, France. E-mail: magnaldo@igr.fr

Received 29 January 2007; Accepted 27 July 2007; Published online 21 August 2007.

Abstract

Stable and safe corrective gene transfer in stem keratinocytes is necessary for ensuring success in cutaneous gene therapy. There have been numerous encouraging preclinical approaches to cutaneous gene therapy in the past decade, but it is only recently that a human volunteer suffering from junctional epidermolysis bullosa could be successfully grafted using his own non-selected, genetically corrected epidermal keratinocytes. However, ex vivo correction of cancer-prone genetic disorders necessitates a totally pure population of stably transduced stem keratinocytes for grafting. Antibiotic selection is not compatible with the need for full respect for natural cell fate potential and avoidance of immunogenic response in vivo. In order to surmount these problems, we developed a strategy for selecting genetically modified stem cell keratinocytes. Driving ectopic expression of CD24 (a marker of post-mitotic keratinocytes) at the surface of clonogenic keratinocytes permitted their full selection. Engineered keratinocytes expressing CD24 and the green fluorescent protein (GFP) tracer gene were shown to retain their original growth and differentiation potentials both in vitro and in vivo over 300 generations. Also, they did not exhibit signs of genetic instability. Using ectopic expression of CD24 as a selective marker of genetically modified human epidermal stem cells appears to be the first realistic approach to safe cutaneous gene therapy in cancer-prone disease conditions.