A preclinical model for the analysis of genetically modified human skin in vivo. 
Although skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer, it is a challenging site when attempting gene delivery.  In addition to the transience of gene expression, important obstacles to cutaneous gene therapy have included the inability to sustain gene expression in a large proportion of keratinocytes within a given skin compartment.  In this study, we have developed a novel experimental strategy that allows long-term regeneration of entirely genetically engineered human skin on the backs of NOD/SCID mice.  Primary human keratinocytes were infected with a retroviral vector encoding the enhanced green fluorescent protein (EGFP) produced by transient transfection of 293T cells.  EGFP expression allowed cell-sorting selection of a polyclonal population of productively transduced keratinocytes that were assembled in a live fibroblast-containing fibrin dermal matrix and orthotopically grafted onto mice.  Epifluorescent illumination of the transplanted zone allowed in vivo monitoring of the genetically modified graft.  EGFP-positive human skin was present on mice for 22 weeks after grafting.  In addition, frozen sections prepared from the grafts displayed consistently strong EGFP-based fluorescence in all epidermal strata at every time point examined.  Persistence of transgene expression was further confirmed through EGFP protein immunodetection.  Purified EGFP-positive keratinocytes grafted as part of the fibrin-based artificial skin were capable of generating multilayer human epidermis on mice, with well-developed granulosum and corneum strata, and clearly defined rete ridges.  Finally, the large proportion of transduced keratinocytes in our grafts allowed us to study, for the first time, the long-term in vivo clonal reconstitution pattern of the regenerated skin.  Analysis of the provirus insertion sites indicates that a discrete number of epidermal stem cell clones was responsible for the maintenance of human skin regenerated in NOD/SCID recipients.