VP22-mediated intercellular transport for suicide gene therapy under oxic and hypoxic conditions

During herpes simplex virus type 1 (HSV 1) infection, the tegument protein VP22 is exported from infected cells to the nuclei of surrounding uninfected cells. These intercellular transport characteristics have prompted the exploitation of VP22 fusion proteins for cancer gene therapy, with the goal of maximizing the bystander effect. Since solid tumors contain hypoxic cell populations that are often refractive to therapy, for efficient targeting, it would be optimal if VP22 functioned even at reduced oxygen concentrations. In the present work, VP22 activity under hypoxic conditions was examined for the first time. Plasmid-transfected human glioma U87-MG and U373-MG cells expressing VP22 fused to the green fluorescent protein (GFP) showed protein export to untransfected cells under tumor oxygenation conditions (0-5% O(2)). For suicide gene therapy, VP22 activity was demonstrated under hypoxia by coupling VP22 to the HSV thymidine kinase (HSVtk). In the presence of the prodrug ganciclovir, cell cultures expressing VP22-HSVtk showed a significant increase in toxicity compared with cells transfected with a construct containing HSVtk only, under all tested conditions. To allow effective suicide gene therapy and simultaneous visualization of therapeutic enzyme localization, a triple fusion protein GFP-HSVtk-VP22 was engineered. Functionality of all components was demonstrated under oxia and hypoxia.