CORRECTIVE TRANSDUCTION OF HUMAN EPIDERMAL STEM-CELLS IN LAMININ-5-DEPENDENT JUNCTIONAL EPIDERMOLYSIS-BULLOSA

Laminin-5 is composed of three distinct polypeptides, alpha 3, beta 3, and gamma 2, which are encoded by three different genes, LAMA3, LAMB3 , and LAMC2, respectively. We have isolated epidermal keratinocytes fr om a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, whi ch led to complete absence of the beta 3 polypeptide. In vitro, beta 3 -null keratinocytes were unable to synthesize laminin-5 and to assembl e hemidesmosomes, maintained the impairment of their adhesive properti es, and displayed a decrease of their colony-forming ability. A retrov iral construct expressing a human beta 3 cDNA was used to transduce pr imary beta 3-null keratinocytes, Clonogenic beta 3-null keratinocytes were transduced with an efficiency of 100%. beta 3-transduced keratino cytes were able to synthesize and secrete mature heterotrimeric lamini n-5, Gene correction fully restored the keratinocyte adhesion machiner y, including the capacity of proper hemidesmosomal assembly, and preve nted 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. Bec ause cultured keratinocytes are used routinely to make autologous graf ts 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 treat ment of genodermatoses.