Defining an innovative and safe non-viral gene delivery system: perspective analysis for gene therapy applications

The Sleeping Beauty (SB) transposon-based integration system is a valuable tool for functional genomics in several model organisms and represent a promising vector for gene therapy in humans. The SB transposase was found to bind the inverted repeats of regenerated salmonid transposons in a substrate-specific manner, and to mediate precise cut-and-paste transposition in fish as well as in mouse and human cells. However, a major bottleneck of any transposon-based application is the efficiency of transpositional activity. Therefore, considerable effort has been made to improve the transposition efficiency of SB by modifying its IRs and systematically mutating the transposase gene. The combined effect of these modifications resulted in the generation of the hyperactive SB100X transposase and of the high-capacity “sandwich” transposon (SA). This new system shows an almost 100-fold enhancement of transposition as compared to the first-generation transposon system. I address my work to the molecular characterization of “sandwich” SB-mediated integrants in epithelial cell lines and in primary keratinocytes. The interest in keratinocytes is related to a SB-mediated gene therapy application for the dystrophic form of epidermolysis bullosa (DEB) caused by mutations in the type-VII collagen gene (COL7A1). Delivering the >9 kb COL7A1 cDNA by a retroviral or lentiviral vector is problematic, due to the large size and highly repeated nature of its sequence, which induce genetic rearrangements during reverse transcription and integration. The Sleeping Beauty transposon-based integration system can potentially overcome these issues.