Twenty years of oncolytic virus development have created a field that is driven by the potential promise of lasting impact on our cancer treatment repertoire. With the field constantly expanding—more than 20 viruses have been recognized as potential oncolytic viruses—new virus candidates continue to emerge even as established viruses reach clinical trials. They all share the defining commonalities of selective replication in tumors, subsequent tumor cell lysis, and dispersion within the tumor. Members from diverse virus classes with distinctly different biologies and host species have been identified. Of these viruses, 15 have been tested on human glioblastoma multiforme. So far, 20 clinical trials have been conducted or initiated using attenuated strains of 7 different oncolytic viruses against glioblastoma multiforme. In this review, we present an overview of viruses that have been developed or considered for glioblastoma multiforme treatment. We outline the principles of tumor targeting and selective viral replication, which include mechanisms of tumor-selective binding, and molecular elements usurping cellular biosynthetic machinery in transformed cells. Results from clinical trials have clearly established the proof of concept and have confirmed the general safety of oncolytic virus application in the brain. The moderate clinical efficacy has not yet matched the promising preclinical lab results; next-generation oncolytic viruses that are either “armed” with therapeutic genes or embedded in a multimodality treatment regimen should enhance the clinical results.

Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma.

Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation.

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Metastatic tumors and malignant gliomas make up the majority of cancers in the brain. They are invariably fatal and there is currently no cure. Fromin vitrocomparisons of a number of viruses, we selected one that appeared the best in selectively killing glioblastoma cells. This replication-competent virus, the glioma-adapted vesicular stomatis virus strain VSVrp30a, was used forin vivotests with the underlying view that infection of tumor cells will lead to an increase in the number of viruses subsequently released to kill additional tumor cells. Intravenous injection of VSVrp30a expressing a green fluorescent protein reporter, rapidly targeted and destroyed multiple types of human and mouse tumors implanted in the mouse brain, including glioblastoma and mammary tumors. When tumors were implanted both in the brain and peripherally, emulating systemic cancer metastasis, tumors inside and outside the brain were simultaneously infected. Intranasal inoculation, leading to olfactory nerve transport of the virus into the brain, selectively infected and killed olfactory bulb tumors. Neither control cortical wounds nor transplanted normal mouse or human cells were targeted, indicating viral tumor selectivity. Control viruses, including pseudorabies, adeno-associated, or replication-deficient VSV, did not infect the brain tumor. Confocal laser time-lapse imaging through a cranial window showed that intravenous VSV infects the tumor at multiple sites and kills migrating tumor cells. Disrupted tumor vasculature, suggested by dye leakage, may be the port of entry for intravenously delivered VSV. Quantitative PCR analysis of how VSVrp30a selectively infected tumor cells suggested multiple mechanisms, including cell surface binding and internalization.

Previously, we described VSV-GP, a modified version of the vesicular stomatitis virus, as a non-neurotoxic oncolytic virus that is effective for the treatment of malignant glioblastoma and ovarian cancer. Here, we evaluate the therapeutic efficacy of VSV-GP for malignant melanoma. All of the human, mouse, and canine melanoma cell lines that were tested, alongside most primary human melanoma cultures, were infected by VSV-GP and efficiently killed. Additionally, we found that VSV-GP prolonged the survival of mice in both a xenograft and a syngeneic mouse model. However, only a few mice survived with long-term tumor remission. When we analyzed the factors that might limit VSV-GP’s efficacy, we found that vector-neutralizing antibodies did not play a role in this context, as even after eight subsequent immunizations and an observation time of 42 weeks, no vector-neutralizing antibodies were induced in VSV-GP immunized mice. In contrast, the type I IFN response might have contributed to the reduced efficacy of the therapy, as both of the cell lines that were used for the mouse models were able to mount a protective IFN response. Nevertheless, early treatment with VSV-GP also reduced the number and size of lung metastases in a syngeneic B16 mouse model. In summary, VSV-GP is a potent candidate for the treatment of malignant melanoma; however, factors limiting the efficacy of the virus need to be further explored.