Vesicular stomatitis virus G pseudotyped retrovector mediates effective invivo suicide gene delivery in experimental brain cancer

Direct in vivo tumor-targeting with "suicide" viral vectors is limited by e ither inefficient gene transfer (ie., retroviral vectors) or indiscriminate transfer of a conditionally toxic gene to surrounding nonmalignant tissue (i.e., adenoviral vectors). Retrovectors pseudotyped with the vesicular sto matitis virus G protein (VSVG) may serve as a remedy to this conundrum. The se retroviral particles differ from standard murine retroviruses by their v ery broad tropism and the capacity to be concentrated by ultracentrifugatio n without loss of activity. We propose that a VSVG-typed retrovector can be used for efficient and tumor-specific herpes simplex virus thymidine kinas e (TK) gene delivery in vivo. To test this hypothesis, we developed a bicis tronic retroviral vector that expresses TI( and green fluorescence protein (pTKiGFP), The 293GPG packaging cell line was used to generate vTKiGFP retr oparticles, In cytotoxicity assays, vTKiGFP-transduced human glioma cell li nes were sensitized to the cytotoxic effects of gangciclovir (GCV) 10,000-f old, Subsequently, virus was concentrated by ultracentrifugation to a titer of 2.3 x 10(10) cfu/ml, We tested the antitumor activity of vTKiGFP retrop articles in a rat C6 glioma model of brain cancer, Concentrated retrovector stock (9 mu l volume) was injected stereotactically in preestablished intr acerebral tumor. Subsequently, rats were treated with GCV for 10 days. Cont rol rats (no GCV) had a mean survival of 38 days (range, 20-52 days), Secti ons performed on postmortem brain tissue revealed large tumors with evidenc e of high efficiency retrovector transfer and expression las assessed by GF P fluorescence). Fluorescence was restricted to malignant tissue. In the ex perimental group (GCV treated), 8 of 12 remain alive and well >120 days aft er glioma implantation, In conclusion, vTKiGFP is very efficient at transdu cing human glioma cell lines in vitro and leads to significant GCV sensitiz ation. Recombinant retroviral particles can he concentrated to titers that allow in vivo intratumoral delivery of large viral doses. The therapeutic e fficiency of this reagent has been demonstrated in a preclinical model of b rain cancer.