Non viral gene transfer via Sleeping beauty transposon for Collagen VII delivery in human primary keratinocytes.

Autosomal recessive epidermolysis bullosa (RDEB) is a genetic skin adhesion defect caused by mutations in the type VII collagen gene (COL7A1). Although full-length type-VII collagen is successfully produced in human keratinocytes by retroviral vectors, genetic instability due to the large size (9kb) and the highly repeated nature of the gene sequence is a persistent problem. The Sleeping-Beauty (SB) transposon-based integration system can potentially overcome these issues by taking advantage of the hyperactive SB100X transposase in combination with the wild-type (pT2) transposon or the “sandwich” version (pSA) that showed robust transposition efficiency in human cells. We molecularly characterized the “sandwich” SB-mediated integrants in epithelial cell lines and in primary keratinocytes. Co-transfecting the transposase together with 10kb-transposon (pT2 or pSA) we observed up to 37% of transposition in HaCaT and in GABEB (generalized atrophic benign epidermolysis bullosa keratinocytes) cells with both transposons. Clonal analysis demonstrated that the transposition events occur with a minimal risk of rearrangements (<3%). LM-PCR based bi-directional sequencing of the transposon-genome junctions shows genuine “cut and paste” activity of the SB hyperactive transposase.