Single-step conversion of cells to retrovirus vector producers with herpessimplex virus-Epstein-Barr virus hybrid amplicons

We report here on the development and characterization of a novel herpes si mplex virus type 1 (HSV-1) amplicon-based vector system which takes advanta ge of the host range and retention properties of HSV-Epstein-Barr virus (EB V) hybrid amplicons to efficiently convert tells to retrovirus sector produ cer cells after single-step transduction, The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequenc e overlap and cloned into an HSV-EBV hybrid amplicon, Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HER E and HERA, which code for the ecotropic and the amphotropic envelopes, res pectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing unit s/ml, while infection of the same cells with amplicon vectors generated max imum titers I order of magnitude lower, Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus ve ctors even at the same transduction efficiencies. Infection of human and do g primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the longterm retention and increase in tr ansgene activity over time in these cell populations. Although the efficien cy of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.