Generation of improved viral hybrid-vectors for stable transduction of mammalian cells

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For a safe and successful gene therapy approach the potential risk of insertional mutagenesis after delivery of integrating viral and non-viral vectors needs to be carefully evaluated. As previously shown for retroviral vectors which predominantly integrate into active genes safety concerns need to be addressed. Although non-viral integrating vectors with a potentially lower risk of insertional mutagenesis were recently developed, one major challenge to be overcome for integrating vectors based on naked DNA is the delivery of the transgene to the target cell and uptake of the recombinant DNA into the cell. To overcome this hurdle the plan of this proposal is to generate novel hybrid-vectors that can efficiently transduce mammalian cells and integrate an expression cassette into the host genome. To accomplish this goal, this proposal will investigate [1] a significantly improved DNA based transposon system in the context of a viral vector and [2] various improved bacteriophage derived DNA integrases within the vector genome for limited integration into the host genome. The final goal [3] of this proposal is to evaluate the relative safety and efficacy of these improved vectors in vitro and in vivo with the primary focus on hematopoietic stem cells[4]. This project will develop novel tools for stable transduction of mammalian cells. It will be an important step towards treating genetic disorders including diseases affecting cells derived from hematopoietic stem cells.