Retrovirus-like particles for protein transfer: A novel technology for transient and stable cell modification

Retroviruses assemble about 5000 copies of Gag precursors which undergo cleavage by the retroviral protease to generate fully processed and bioactive retroviral proteins. We hypothesized that this process might be exploited for delivery of foreign proteins into target cells after incorporation of the protein of choice into Gag. We demonstrate that murine leukemia virus (MLV) tolerates fusion of green fluorescent protein (GFP) at different positions within Gag. Kinetic analysis showed the transient behavior of the system and revealed the presence of the transferred GFP up to 4 days. When GFP was separated from its retroviral fusion partner by an additional protease cleavage site, fluorescence microscopy showed its localization in the cytoplasm of target cells while released nlsGFP (carrying a nuclear localization signal) translocated towards the nucleus. Encouraged by these results we engineered retroviral particles that deliver a bioactive protein, the site specific recombinase Flp. Using fibroblasts carrying a Flp-recombination dependent indicator construct we show that Flp can be efficiently delivered in a dose-controlled and receptor-mediated manner. In order to test the technology in a relevant cell type, we engineered induced pluripotent stem (iPS) cells carrying the same Flp-indicator construct and transduced them with our retroviral particles delivering Flp. Importantly, fluorescence microscopy and flow cytometry revealed an efficient Flp-mediated recombination in iPS cells. Finally, we generated "bipotent" retroviral Flp-transducing particles to co-transfer nucleic acids. This opens the door for a variety of different applications in basic research and biomedicine.