Transplantation of autologous, genetically corrected epidermal stem cells (EpSC) was successfully used to treat junctional epidermolysis bullosa (EB), a genetic skin adhesion disorder. The dystrophic forms of EB is caused by mutations in the type-VII collagen gene (COL7A1) Delivering the >9 kb COL7A1 cDNA by a retroviral vector is problematic, due to the large size and highly repeated nature of its sequence, which induce genetic rearrangements during reverse transcription and integration. We tested the feasibility of using a non-viral vector system based on Sleeping Beauty (SB)-derived transposons, taking advantages of the recently developed, high-capacity “sandwich” version of the SB transposon and the “hyperactive” SB 100X transposase, which showed high transposition efficiency in human stem cells. We tested the system in HeLa cells and in a keratinocyte cell line (HaCaT), which were co-transfected with the SB 100X transposase and either the normal or the sandwich version of the SB transposon containing a small-size reporter gene (Venus or GFP) expression cassette. In both cell lines, transposition was obtained in up to 80% of the transfected cells with the sandwich transposon, compared to ~50% obtained with the older version. Transposed HaCaT cells were cloned and analysed for integration events. Individual clones carried several copies of the integrated transposon of the predicted size. High-throughput sequencing is under way to analyze the sandwich SB transposon integration characteristics. We then tested a transposon carrying an 8.8-kb cassette, which again showed up to 80% transposition efficiency in transfected cells. Finally, we generated sandwich transposons containing an expression cassette for the COL7A1 cDNA under the control of a constitutive (PGK) or a keratinocyte-specific (K14) promoter, which are currently being tested for integration in HaCaT cells. The SB-based gene delivery system will finally be tested in human primary keratinocyte cultures.
Development of a Sleeping Beauty transposon-based integration system for gene transfer in human epithelial cells / Turchiano, Giandomenico; Latella, Maria Carmela; Izsvak, Zsuzsanna; Ivics, Zoltan; Mavilio, Fulvio; Recchia, Alessandra. - ELETTRONICO. - 1:(2011), pp. 1-1. (Intervento presentato al convegno ASGCT meeting tenutosi a S nel 18-22 maggio 2011).