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Laminin-5 is composed of three distinct polypeptides, α3, β3, and γ2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the β3 polypeptide. In vitro, β3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human β3 cDNA was used to transduce primary β3-null keratinocytes. Clonogenic β3-null keratinocytes were transduced with an efficiency of 100%. β3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses. Epidermolysis bullosa (EB) is an heritable devastating blistering disorder of the skin that is due to mutations in genes encoding several structural proteins, as keratins (simplex EB), laminin-5, and its integrin α6 β4 receptor (junctional EB), type VII collagen (dystrophic EB). Here we show that a LAMB3-dependent junctional EB can be stably corrected in vitro through retrovirus-mediated transduction of epidermal stem cells. Autologous cultured keratinocytes are used for the permanent coverage of massive full-thickness burns or in the restoration of severely damaged lining epithelia. Therefore, the possibility of stable corrective transduction of epidermal stem cells indicates that keratinocyte-mediated ex vivo gene therapy of skin genetic diseases can be feasible.
Dellambra et al. (Wed,) studied this question.