Samuel D. Rabkin

Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial

G207 is a conditionally replicating derivative of herpes simplex virus (HSV) type-1 strain F engineered with deletions of both γ134.5 loci and a lacZ insertion disabling the UL39 gene. We have demonstrated the efficacy of G207 in treating malignant glial tumors in athymic mice, as well as the safety of intracerebral G207 inoculation in mice and in Aotus nancymai. We sought to determine the safety of G207 inoculation into cerebral malignant glial tumors in humans. Criteria for inclusion into this dose-escalation study were the diagnosis of histologically proven malignant glioma, Karnofsky score ⩾70, recurrence despite surgery and radiation therapy, and an enhancing lesion greater than 1 cm in diameter. Serial magnetic resonance images were obtained for volumetric analysis. The trial commenced at a dose of 106 plaque forming units (p.f.u.) inoculated at a single enhancing site and was completed when the 21st patient was inoculated with 3 × 109 p.f.u. at five sites. While adverse events were noted in some patients, no toxicity or serious adverse events could unequivocally be ascribed to G207. No patient developed HSV encephalitis. We found radiographic and neuropathologic evidence suggestive of anti-tumor activity and long-term presence of viral DNA in some cases.

Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas.

We have created a double mutant of the herpes simplex virus (HSV) type 1 (termed G207) with favourable properties for treating human malignant brain tumours: replication–competence in glioblastoma cells (and other dividing cells), attenuated neurovirulence, temperature sensitivity, ganciclovir hypersensitivity, and the presence of an easily detectable histochemical marker. G207 has deletions at both γ34.5 (RL1) loci and a lacZ gene insertion inactivating the ICP6 gene (UL39). G207 kills human glioma cells in monolayer cultures. In nude mice harbouring subcutaneous or intracerebral U–87MG gliomas, intraneoplastic inoculation with G207 causes decreased tumour growth and/or prolonged survival. G207 is avirulent upon intracerebral inoculation of mice and HSV–sensitive non–human primates. These results suggest that G207 should be considered for clinical evaluation in the treatment of glioblastomas.

Analgesia and hyperalgesia from GABA-mediated modulation of the cerebral cortex.

It is known that pain perception can be altered by mood, attention and cognition, or by direct stimulation of the cerebral cortex, but we know little of the neural mechanisms underlying the cortical modulation of pain. One of the few cortical areas consistently activated by painful stimuli is the rostral agranular insular cortex (RAIC) where, as in other parts of the cortex, the neurotransmitter γ-aminobutyric acid (GABA) robustly inhibits neuronal activity. Here we show that changes in GABA neurotransmission in the RAIC can raise or lower the pain threshold—producing analgesia or hyperalgesia, respectively—in freely moving rats. Locally increasing GABA, by using an enzyme inhibitor or gene transfer mediated by a viral vector, produces lasting analgesia by enhancing the descending inhibition of spinal nociceptive neurons. Selectively activating GABAB-receptor-bearing RAIC neurons produces hyperalgesia through projections to the amygdala, an area involved in pain and fear. Whereas most studies focus on the role of the cerebral cortex as the end point of nociceptive processing, we suggest that cerebral cortex activity can change the set-point of pain threshold in a top-down manner.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Human Glioblastoma–Derived Cancer Stem Cells: Establishment of Invasive Glioma Models and Treatment with Oncolytic Herpes Simplex Virus Vectors

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.

Oncolytic herpes simplex virus vectors for cancer virotherapy

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are emerging as an effective and powerful therapeutic approach for cancer. Replication-competent HSV-1 vectors with mutations in genes that affect viral replication, neuropathogenicity, and immune evasiveness have been developed and tested for their safety and efficacy in a variety of mouse models. Evidence to-date following administration into the brain attests to their safety, an important observation in light of the neuropathogenicity of the virus. Phase I clinical traits of three vectors, G207, 1716, and NV1020, are either ongoing or completed, with no adverse events attributed to the virus. These and other HSV-1 vectors are effective against a myriad of solid tumors in mice, including glioma, melanoma, breast, prostate, colon, ovarian, and pancreatic cancer. Enhancement of activity was observed when HSV-1 vectors were used in combination with traditional therapies such as radiotherapy and chemotherapy, providing an attractive strategy to pursue in the clinic. Oncolytic HSV-1 vectors expressing “suicide” genes (thymidine kinase, cytosine deaminase, rat cytochrome P450) or immunostimulatory genes (IL-12, GM-CSF, etc.) have been constructed to maximize tumor destruction through multimodal therapeutic mechanisms. Further advances in virus delivery and tumor specificity should improve the likelihood for successful translation to the clinic.

Systemic Antitumor Immunity in Experimental Brain Tumor Therapy Using a Multimutated, Replication-Competent Herpes Simplex Virus

Replication-competent, attenuated herpes simplex virus (HSV) vectors have been developed for viral oncolytic therapy of primary and metastatic malignant brain tumors. However, the role of the host immune responses in the brain has not been elucidated. N18 neuroblastoma cells were used as a tumor model in syngeneic A/J mice to test the therapeutic efficacy of G207, a conditionally replicating HSV vector, in an immunocompetent condition. G207 inoculated intraneoplastically exhibited a prominent oncolytic antitumor effect in mice harboring N18 tumors in the brain or subcutaneously, and, in addition, elicited a systemic antitumor immune response. Subcutaneous tumor therapy with G207 caused regression of a remote, established tumor in the brain or in the periphery, which was potentially mediated by the systemic antitumor immune response, and provided persistent tumor-specific protection against N18 tumor rechallenge in the brain as well as in the periphery. Antitumor immunity was associated with an elevation of specific CTL activity against N18 tumor cells that persisted for at least 13 months. The results suggest that the oncolytic antitumor action of replication-competent HSV may be augmented by induction of specific and systemic antitumor immunity effective both in the periphery and in the brain.

Local and systemic therapy of human prostate adenocarcinoma with the conditionally replicating herpes simplex virus vector G207

Prostate adenocarcinoma is the most common nonskin malignancy in males and the second most common cause of cancer death in the United States (Landis et al., 1998). Initial treatments of surgery or radiotherapy may cause impotence and/or incontinence from neural damage (Eastham and Scardino, 1998; Porter et al., 1998). When extraprostatic or metastatic disease develops, castration or pharmaceutical androgen ablation is utilized (Catalona, 1994). Androgen-resistant recurrence indicates a poor prognosis and justifies experimental chemotherapy (Oh and Kantoff, 1998). G207 (Mineta et al., 1995; Yazaki et al., 1995) is a multimutated herpes simplex virus 1 (HSV) vector that replicates within cancer cells, causing cellular death; however, replication is limited in normal cells, including those of the nervous system. In vitro, G207 at a low multiplicity of infection (MOI of 0.01) is oncolytic for multiple human prostate cancer cells. In athymic mice, a single intraneoplastic inoculation of G207 completely eradicates >22% of established subcutaneous human prostate cancer tumors irrespective of hormonal responsiveness. Two intraneoplastic inoculations of G207 completely eradicated two of three recurrent previously irradiated tumors and two intravenous administration of G207 induced tumor regression in distant subcutaneous tumors and completely eradicated one-fourth of the tumors.

Expression of a functional foreign gene in adult mammalian brain following in Vivo transfer via a herpes simplex virus type 1 defective viral vector

We report here the first use of a herpes simplex virus defective viral vector for the transfer and expression of a foreign gene in the adult rat brain in vivo. Defective vectors offer unique advantages over other systems. Our vector genome consists of multiple copies of a plasmid-based amplicon, with a human cytomegalovirus promoter and lacZ gene as a reporter. Helper functions were provided by an HSV1 mutant incapable of replication at physiological temperatures. The resulting defective viral vector was stereotaxically microinjected into rat hippocampus and hypothalamus, and cells expressing functional beta-galactosidase were detected in both areas. Expression was confined to regions at or near the site of injection. Positive cells were identified by 18 hr following injection, and expression was still detectable after 2 weeks. All animals survived with no behavioral, gross, or microscopic anatomical evidence of a virulent neurologic infection.

Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells

The clinicopathological heterogeneity of glioblastoma (GBM) and the various genetic and phenotypic subtypes in GBM stem cells (GSCs) are well described. However, the relationship between GSCs and the corresponding primary tumor from which they were isolated is poorly understood. We have established GSC-enriched neurosphere cultures from 15 newly diagnosed GBM specimens and examined the relationship between the histopathological and genomic features of GSC-derived orthotopic xenografts and those of the respective patient tumors. GSC-initiated xenografts recapitulate the distinctive cytological hallmarks and diverse histological variants associated with the corresponding patient GBM, including giant cell and gemistocytic GBM, and primitive neuroectodermal tumor (PNET)-like components. This indicates that GSCs generate tumors that preserve patient-specific disease phenotypes. The majority of GSC-derived intracerebral xenografts (11 of 15) demonstrated a highly invasive behavior crossing the midline, whereas the remainder formed discrete nodular and vascular masses. In some cases, GSC invasiveness correlated with preoperative MRI, but not with the status of PI3-kinase/Akt pathways or O(6)-methylguanine methyltransferase expression. Genome-wide screening by array comparative genomic hybridization and fluorescence in situ hybridization revealed that GSCs harbor unique genetic copy number aberrations. GSCs acquiring amplifications of the myc family genes represent only a minority of tumor cells within the original patient tumors. Thus, GSCs are a genetically distinct subpopulation of neoplastic cells within a GBM. These studies highlight the value of GSCs for preclinical modeling of clinically relevant, patient-specific GBM and, thus, pave the way for testing novel anti-GSC/GBM agents for personalized therapy.