Assessing and improving the safety profile of Sleeping Beauty transposon-mediated gene transfer in human cells

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Transposons are natural gene delivery vehicles. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term expression of transgenes in cells of a wide range of vertebrates, including humans. SB transposition, during which the element ¿jumps¿ from one DNA molecule to another, occurs into human chromosomal DNA in a fairly random manner. Thus, SB has a mutagenic potential, because it can insertionally activate or inactivate genes. This is useful for gene discovery in vertebrate models, but is clearly undesired for human gene therapeutic applications. In this project, we will determine the effects of transposase expression and transposon integration on cellular processes. We propose to investigate the molecular mechanisms involved in SB¿s target site selection, by mutagenizing the region of the SB transposase responsible for target DNA capture. Mutants will be analyzed for altered function on the levels of primary DNA binding, transposon excision and transposon integration in human cells. We further propose to manipulate SB¿s target site selection in order to achieve targeted transposition into predetermined loci or chromosomal regions. We consider experimental strategies based on targeting fusion proteins composed of a specific DNAbinding protein domain, responsible for binding to chromosomal DNA, and either the transposase polypeptide or another protein that interacts with the transposase or with the transposon DNA. Insights into the molecular mechanisms involved in target site selection of transposable elements can lead to powerful methods for safe transgene integration in human applications, and for target-selected gene knock-outs in vertebrate models.