Transposable elements are non-viral gene delivery vehicles found ubiquitously in nature. Transposon-based vectors have the capacity of stable genomic integration and long-lasting expression of transgene constructs in cells. Molecular reconstruction of Sleeping Beauty (SB), an ancient transposon in fish, represents a milestone in applying transposition-mediated gene delivery in vertebrate species, including humans. SB appears to be a safer vector for therapeutic gene delivery than most virus-based integrating vector systems, because it does not display a preference for integration into (expressed) genes. Nevertheless, SB has a mutagenic potential, because it can insertionally activate or inactivate genes as a result of random genomic transposition. In this project, we will investigate target site preferences of SB by large-scale, next generation sequencing of SB transposon insertion sites recovered from human cells. This analysis will provide a thus far unprecedented resolution of genomic sites accessible to transposable elements. We will further determine the effects of transposon integration on cellular processes, and thereby assess the genotoxic potential of transposon vectors in human cells. Finally, we propose to manipulate SB's target site selection in order to achieve targeted transposition into physiologically relevant loci in the human genome. Targeted integration of a therapeutic gene to a "safe" site in the genome would significantly improve the safety profile of transposition-mediated gene transfer in human cells.

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