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Background: Glioblastoma is a fatal brain tumor in needing urgent effective therapy. Treatments with both oncolytic viruses and immunotherapy have shown preclinical efficacy and clinical promise. We sought to exploit possible synergies between oncolytic herpes simplex virus type 1 (oHSV-1) infection of intracranial gliomas and delivery of immune-stimulating fms-like tyrosine kinase 3 ligand (Flt3L) by engineering a herpes vector to express the cytokine., Objective: To construct an oHSV-1 vector that expresses high levels of Flt3L and examine its antiglioma efficacy in an immunocompetent murine model., Methods: G47Δ and a bacterial artificial chromosome system were used to generate a novel oHSV-1, termed G47Δ-Flt3L, expressing Flt3L. Cytokine expression was confirmed, and G47Δ-Flt3L was injected intratumorally into established intracranial CT-2A gliomas in syngeneic C57/Bl6 mice. Animals were followed for survival and assessed by the Kaplan-Meier method., Results: G47Δ-Flt3L expressed high levels of Flt3L in culture. Expression of Flt3L affected neither viral replication nor had a cytotoxic effect on CT2A glioma cells. Direct inoculation into intracerebral CT2A glioma cells resulted in high levels of detectable Flt3L in mouse blood and was superior to parental G47Δ in prolonging survival in glioma-bearing animals., Conclusion: Treatment with G47Δ-Flt3L improves survival of glioma-bearing mice.

The ICP34.5 protein of herpes simplex virus (HSV) is involved in many aspects of viral pathogenesis; promoting neurovirulence, inhibiting interferon-induced shutoff of protein synthesis, interacting with PCNA and TBK1, inhibiting dendritic cell (DC) maturation, and binding to Beclin 1 to interfere with autophagy. Because of its key role in neuropathogenicity, the γ34.5 gene is deleted in all oncolytic HSVs (oHSVs) currently in clinical trial for treating malignant gliomas. Unfortunately, deletion of γ34.5 attenuates virus replication in cancer cells, especially human glioblastoma stem cells (GSCs). To develop new oHSVs for use in the brain and that replicate in GSCs, we explored the effect of deleting the γ34.5 Beclin 1 binding domain (BBD). To ensure cancer selectivity and safety, we inactivated the ICP6 gene (UL39, large subunit of ribonucleotide reductase), constructing ICP6 mutants with different γ34.5 genotypes: Δ68HR-6, intact γ34.5; Δ68H-6, γ34.5 BBD deleted; and 1716-6, γ34.5 deleted. Multimutated Δ68H-6 exhibited minimal neuropathogenicity in HSV-1-susceptible mice, as opposed to Δ68H and Δ68HR-6. It replicated well in human glioma cell lines and GSCs, effectively killing cells in vitro and prolonging survival of mice bearing orthotopic brain tumors. In contrast, 1716 and 1716-6 barely replicated in GSCs. Infection of glioma cells with Δ68H-6 and 1716-6 induced autophagy and increased phosphorylation of eIF2α, while inhibition of autophagy, by Beclin 1 short hairpin RNA (shRNA) knockdown or pharmacological inhibition, had no effect on virus replication or phosphorylated eIF2α (p-eIF2α) levels. Thus, Δ68H-6 represents a new oHSV vector that is safe and effective against a variety of brain tumor models.

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.