Transplantation of autologous, genetically corrected epidermal stem cells (EpSC) is a potential treatment for junctional epidermolysis bullosa, a genetic skin adhesion disorder. Targeted transgene integration overcomes the issue of random insertional mutagenesis associated with retroviral vectors, and may thus provides a safer gene transfer alternative. We developed a gene-targeting platform based on the use of zinc-finger nucleases (ZFNs) and integrase-defective lentiviral vectors (IDLVs) to insert a transgene by homologous recombination (HR) into the AAVS1 locus on chromosome 19. We evaluated the targeting efficiency in a keratinocyte cell line (HaCaT) by IDLV-mediated delivery of an AAVS1-specific ZFN pair together with an HR construct driving the insertion of a GFP expression cassette into the site of cleavage. We achieved up to 25% of targeted insertion of single copies or concatamers of the GFP cassette into the AAVS1 locus, as analyzed by PCR, Southern blotting and sequencing on individual HaCaT cell clones. Evidence of HR-mediated targeted integration was also obtained at a lower but significant frequency in human primary keratinocyte cultures, by using ZFNs-expressing Adeno vector together with IDLV carrying the GFP expression cassette flanked by AAVS1 homology arms. We observed up to 9% disruption of the ZFN target site, repaired by non-homologous end joining, as evaluated by Cel-1 assay and pyrosequencing. These data suggest that the major limitation of ZFN-mediated targeted integration is represented by poor induction of the HR-dependent DNA repair pathway. To study gene targeting in repopulating keratinocyte stem cells, human skin equivalents derived from keratinocytes co-infected with IDLV donor and AdZFNs vectors were grafted onto immunodeficient (nu/nu) mice. GFP-positive spots were observed for at least 10 weeks in the grafted tissue, confirming that stable integration occurred in transplantable keratinocyte stem cells.
Targeted gene integration in human epidermal stem cells by Zinc-finger nuclease-mediated homologous recombination / Coluccio, Andrea; Lombardo, Angelo; Miselli, Francesca; Holmes, Michael; Gregory, Philip D.; Naldini, Luigi; Recchia, Alessandra; Mavilio, Fulvio. - ELETTRONICO. - 1:(2012), pp. 1-1. (Intervento presentato al convegno ASGCT meeting tenutosi a Philadelphia nel 16-20 maggio 2012).