The combination of G47Δ and TMZ acts synergistically in killing GSCs through oHSV-mediated manipulation of DNA damage responses. This strategy is highly efficacious in representative preclinical models and warrants clinical translation.

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 ␥34.5 gene is deleted in all oncolytic HSVs (oHSVs) currently in clinical trial for treating malignant gliomas. Unfortunately, deletion of ␥34.5 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 ␥34.5 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 ␥34.5 genotypes: ⌬68HR-6, intact ␥34.5; ⌬68H-6, ␥34.5 BBD deleted; and 1716-6, ␥34.5 deleted. Multimutated ⌬68H-6 exhibited minimal neuropathogenicity in HSV-1-susceptible mice, as opposed to ⌬68H and ⌬68HR-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 ⌬68H-6 and 1716-6 induced autophagy and increased phosphorylation of eIF2␣, while inhibition of autophagy, by Beclin 1 short hairpin RNA (shRNA) knockdown or pharmacological inhibition, had no effect on virus replication or phosphorylated eIF2␣ (p-eIF2␣) levels. Thus, ⌬68H-6 represents a new oHSV vector that is safe and effective against a variety of brain tumor models.

In the adult human brain, the presence of neural stem cells has been documented in the subgranular layer of the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles. Neurogenesis has also been reported in rodent models of ischemic stroke, traumatic brain injury, epileptic seizures, and intracerebral or subarachnoid hemorrhage. However, only sparse information is available about the occurrence of neurogenesis in the human brain under similar pathological conditions. In the present report, we describe neural progenitor cell proliferation in the brain of patients suffering from subarachnoid hemorrhage (SAH) resulting from ruptured aneurysm. Ten cerebral samples from both SAH and control patients obtained, respectively, during aneurysm clipping and deep brain tumor removal were analyzed by reverse transcription followed by polymerase chain reaction (RT-PCR) and/or immunohistochemistry (IHC). In tissue specimens from SAH patients, RT-PCR and IHC revealed the expression of a variety of markers consistent with CNS progenitor cells, including nestin, vimentin, SOX-2, and Musashi1 and -2. In the same specimens, double immunohistochemistry followed by confocal analysis revealed that Musashi2 consistently colocalized with the proliferation marker Ki67. By contrast, no such gene or protein expression profiles were detected in any of the control specimens. Thus, activation of neural progenitor cell proliferation may occur in adult human brain following subarachnoid hemorrhage, possibly contributing to the promotion of spontaneous recovery, in this pathological condition.

Glioblastoma (GBM), a fatal malignant brain tumor, contains abundant hypoxic regions that provide a "niche" to promote both the maintenance and enrichment of glioblastoma stem-like cells (GSCs) and confer resistance to chemo-and radiotherapy. Since GSCs, with an ability to resist conventional therapies, may be responsible for tumor recurrence, targeting GSCs located in such a hypoxic environment may be critical to improving the therapeutic outcome for GBM patients. Oncolytic viral therapies have been tested in the clinic as a promising therapeutic approach for GBM. In this study, we analyzed and compared the therapeutic effects of oncolytic herpes simplex virus (oHSV) type 1 G47⌬ (␥34.5 − ICP6 − LacZ ؉ ␣47 − ) in patient-derived GSCs under normoxia (21% oxygen) and hypoxia (1% oxygen). GSCs cultured in hypoxia showed an increased ability to form neurospheres and expressed higher levels of the putative stem cell marker CD133 compared with GSCs cultured in normoxia. G47⌬ exhibited a comparable ability to infect, replicate, and kill GSCs in normoxia and hypoxia in vitro. Importantly, G47⌬ could counteract hypoxia-mediated enhancement of the stemlike properties of GSCs, inhibiting their self-renewal and stem cell marker expression. Using orthotopic human GSC xenografts in mice, we demonstrated that intratumoral injection of G47⌬Us11fluc, a newly developed G47⌬ derivative that expresses firefly luciferase driven by a true late viral promoter, led to an equivalent frequency of viral infection and replication in hypoxic and nonhypoxic tumor areas. These findings suggest that oHSV G47⌬ represents a promising therapeutic strategy to target and kill GSCs, not only in normoxic areas of GBM but also within the hypoxic niche. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:322-332