Efficient gene transfer to human epidermal keratinocytes on fibronectin:: In vitro evidence for transduction of epidermal stem cells

The epidermis is an attractive target for gene therapy because it is easily accessible and has great potential as an ectopic site for protein delivery in vivo. Genetically modified keratinocytes can be expanded in culture and used to generate three-dimensional skin equivalents, which can deliver therapeutic proteins either locally or systemically for the treatment of wounds or systemic diseases. Here we present an optimum protocol that yields consistently high retroviral gene transfer on a substrate of recombinant fibronectin (rFN). Gene transfer on rFN depends strongly on virus concentration and the density of target cells. Interestingly, the kinetics of gene transfer varies depending upon the origin-mouse or human-of virus-producer cells. Most notably, long-term growth and clonogenic assays show that transduction on rFN promotes gene transfer to epidermal stem cells and prevents loss of clonogenic potential due to exposure of cells to retroviral supernatant. In contrast, the traditional protocol transduces mostly differentiated keratinocytes. We also show that skin equivalents prepared from genetically modified keratinocytes display high levels of transgene expression, mainly in the suprabasal layers. Our results are important for cutaneous gene therapy and for biological studies that require efficient and permanent genetic modification.