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.
Del Rio
Marcela
M
Project for Cell and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, 28040 Madrid, Spain. marcela.delrio@ciemat.es
Larcher
Fernando
F
Serrano
Fernando
F
Meana
Alvaro
A
Muñoz
Marta
M
Garcia
Marta
M
Muñoz
Evangelina
E
Martin
Clara
C
Bernad
Antonio
A
Jorcano
José Luis
JL
D000818:Animals; D015139:Blotting, Southern; D004817:Epidermis / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D012038:Regeneration; D012190:Retroviridae / Q000235:genetics*; D012867:Skin / Q000166:cytology / Q000378:metabolism*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005337:Fibrin