New approaches towards ex vivo and in vivo gene therapy

A number of hurdles have to be overcome for efficient and specific gene therapy approaches. Here, we report on two different strategies that should lead to an improvement of current protocols. A strategy is presented to tag unique chromosomal integration sites by means of retroviral infection, which can be reused for exchange with the gene of interest by action of site-specific recombinases. Targeting exchange is achieved in one step with 100% efficiency by a stringent positive selection, which makes further screening superfluous. With this strategy a predictable gene expression is obtained for foreign genes integrated into a predefined chromatin structure. A second approach aims at the stabilization of mouse retroviruses towards human serum which is a prerequisite for in vivo gene therapy protocols. To stabilize murine leukemia virus-based retroviruses against human serum, complement regulatory proteins were fused to the retroviral ENV proteins. This resulted in infectious and human complement-protected particles.