We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.

We have recently shown that vesicular stomatitis virus (VSV) exhibits potent oncolytic activity both in vitro and in vivo (S. Balachandran and G. N. Barber, IUBMB Life 50: [135][136][137][138] 2000). In this study, we further demonstrated, in vivo, the efficacy of VSV antitumor action by showing that tumors that are defective in p53 function or transformed with myc or activated ras are also susceptible to viral cytolysis. The mechanism of viral oncolytic activity involved the induction of multiple caspase-dependent apoptotic pathways was effective in the absence of any significant cytotoxic T-lymphocyte response, and occurred despite normal PKR activity and eIF2␣ phosphorylation. In addition, VSV caused significant inhibition of tumor growth when administered intravenously in immunocompetent hosts. Our data indicate that VSV shows significant promise as an effective oncolytic agent against a wide variety of malignant diseases that harbor a diversity of genetic defects.Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus and prototypic member of the family Rhabdoviridae that is extremely sensitive to the antiviral actions of the interferons (IFNs), a family of cytokines produced in response to viral infection that act by inducing the expression of more than 30 genes (9, 14). Indeed, the importance of the IFNs in controlling VSV infection has been underscored by research demonstrating that embryonic fibroblasts and mice lacking a functional IFN system or the IFN-inducible double-stranded RNAdependent protein kinase PKR are extremely susceptible to VSV infection (2, 6a, 7a, 7b, 11). In addition to these studies, it has become apparent that although VSV replicates inefficiently in primary cells that contain a functional IFN/PKR system, this virus can replicate to high titers in a majority of immortalized and transformed tissue culture cell lines. It currently remains unclear whether aspects of IFN signaling and PKR action may be compromised in such malignant cells, thus affording a cellular environment that would facilitate viral replication. Nevertheless, we and others recently exploited these observations and demonstrated that VSV could also selectively inhibit, in vivo, the growth of tumors derived from transformed cells (1, 12). Our findings indicate that VSV could provide a potentially novel antitumor therapy.Rat C6 glioblastoma cells are permissive to VSV. As a start to analyzing the mechanisms of VSV-induced oncolysis, we examined the kinetics of VSV replication in the rat p53-defective C6 glioblastoma (C6) cell line (4). We selected this cell line for further study because we had previously shown that VSV causes the potent inhibition of C6-derived tumor growth in athymic nude mice (1). Accordingly, C6 cells were infected with VSV (multiplicity of infection [MOI] ϭ 1), and lysates, prepared at various time points postinfection (p.i.), were analyzed for viral replication by immunoblotting for VSV proteins. As shown in Fig. 1A, viral protein synthesis was readily detectable within 24 h of infection...

The rSCC-61/SCC-61 model provides the opportunity for future investigations of the redox-regulated mechanisms of response to combined radiation and Erlotinib in a preclinical setting.