Jacqueline N. Parker

Phase Ib Trial of Mutant Herpes Simplex Virus G207 Inoculated Pre-and Post-tumor Resection for Recurrent GBM

We have previously demonstrated safety of G207, a doubly mutated (deletion of both gamma(1)34.5 loci, insertional inactivation of U(L)39) herpes simplex virus (HSV) for patients stereotactically inoculated in enhancing portions of recurrent malignant gliomas. We have now determined safety of two inoculations of G207, before and after tumor resection. Inclusion criteria were histologically proven recurrent malignant glioma, Karnofsky score >or=70, and ability to resect the tumor without ventricular system breach. Patients received two doses of G207 totaling 1.15 x 10(9) plaque-forming units with 13% of this total injected via a catheter placed stereotactically in the tumor. Two or five days later, tumor was resected en bloc with catheter in place. The balance of G207 dose was injected into brain surrounding the resection cavity. Six patients with recurrent glioblastoma multiforme were enrolled. Two days after the second G207 inoculation, one patient experienced transient fever, delirium, and hemiparesis, which entirely resolved on high-dose dexamethasone. No patient developed HSV encephalitis or required treatment with acyclovir. Radiographic and neuropathologic evidence suggestive of antitumor activity is reported. Evidence of viral replication was demonstrated. G207 appears safe for multiple dose delivery, including direct inoculation into the brain surrounding tumor resection cavity.

Increased efficacy of an interleukin-12-secreting herpes simplex virus in a syngeneic intracranial murine glioma model

Long-term survivors of glioblastoma multiforme, the most common form of primary intracranial malignancy in adults, are extremely rare. Experimental animal models that more closely resemble human disease are essential for the identification of effective novel therapies. We report here an extensive analysis of the 4C8 glioma model to assess its suitability for evaluating novel type 1 herpes simplex virus (HSV-1) therapies of malignant glioma. We first determined that expression of major histocompatibility complex I and II and of alphavbeta3 in the 4C8 model was comparable to that seen in human glioma cells. Next, using a panel of Delta(gamma1)34.5 HSVs, we demonstrated that, in vitro, 4C8 cells were as sensitive as human glioma cells to both infection and lysis and that the 4C8 cells supported the production of foreign gene products. Replication competence of HSV was demonstrated in vitro. Finally, 4C8 intracranial gliomas were established in immunologically competent syngeneic B6D2F1 mice, treated by intratumoral injection of selected engineered HSVs, including the interleukin-12-expressing virus, M002. Survival data from these studies demonstrated that 4C8 cells in vivo are sensitive to both direct oncolysis and HSV-mediated interleukin-12 expression. Fluorescence-activated cell sorting analyses of immune-related infiltrating cells supported the concept that survival was prolonged in part because of antitumor actions of these cells. We conclude that the 4C8/B6D2F1 syngeneic glioma model is suitable for preclinical evaluation of HSV-based therapies and that M002 is a superior virus for the treatment of murine glioma in this model.

Oncolytic viral therapy of malignant glioma.

SummaryNovel approaches to treatment of malignant glioma, the most frequently occurring primary brain tumor, have included the use of a wide range of oncolytic viral vectors. These vectors, either naturally tumor-selective, or engineered as such, have shown promise in the handful of phase I and phase II clinical trials conducted in recent years. The strategies developed for each of the different viruses currently being studied and the history of their development are summarized here. In addition, the results of clinical trials in patients and their implication for future trials are also discussed.

A Phase 1 Trial of Oncolytic HSV-1, G207, Given in Combination With Radiation for Recurrent GBM Demonstrates Safety and Radiographic Responses

G207, a mutant herpes simplex virus (HSV) type 1, is safe when inoculated into recurrent malignant glioma. We conducted a phase 1 trial of G207 to demonstrate the safety of stereotactic intratumoral administration when given 24 hours prior to a single 5 Gy radiation dose in patients with recurrent malignant glioma. Nine patients with progressive, recurrent malignant glioma despite standard therapy were included. Patients received one dose of G207 stereotactically inoculated into the multiple sites of the enhancing tumor margin and were then treated focally with 5 Gy radiation. Treatment was well tolerated, and no patient developed HSV encephalitis. The median interval between initial diagnosis and G207 inoculation was 18 months (mean: 23 months; range: 11-51 months). Six of the nine patients had stable disease or partial response for at least one time point. Three instances of marked radiographic response to treatment occurred. The median survival time from G207 inoculation until death was 7.5 months (95% confidence interval: 3.0-12.7). In conclusion, this study showed the safety and the potential for clinical response of single-dose oncolytic HSV therapy augmented with radiation in the treatment of malignant glioma patients. Additional studies with oncolytic HSV such as G207 in the treatment of human glioma are recommended.

Posttranscriptional control of type I interferon genes by KSRP in the innate immune response against viral infection.

ABSTRACT Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3′ untranslated regions. Expression of ARE-containing type I interferon transcripts is robustly induced upon viral infection and rapidly shut off thereafter. Their transient accumulation is partly mediated through posttranscriptional regulation. Here we show that mouse embryonic fibroblasts derived from knockout mice deficient in KH-type splicing regulatory protein (KSRP), an RNA-binding protein required for ARE-mediated mRNA decay, produce higher levels of Ifna and Ifnb mRNAs in response to viral infection as a result of decreased mRNA decay. Functional analysis showed that KSRP is required for the decay of Ifna4 and Ifnb mRNAs by interaction with AREs. The increased IFN expression renders Ksrp−/− cells refractory to herpes simplex virus type 1 and vesicular stomatitis virus infection. These findings support a role of a posttranscriptional mechanism in the control of type I IFN expression and highlight the function of KSRP in innate immunity by negatively regulating IFN production.

Engineered herpes simplex virus expressing bacterial cytosine deaminase for experimental therapy of brain tumors

Lack of effective therapy of primary brain tumors has promoted the development of novel experimental approaches utilizing oncolytic viruses combined with gene therapy. Towards this end, we have assessed a conditionally replication-competent, γ134.5-deleted herpes simplex virus type 1 (HSV-1) expressing cytosine deaminase (CD) for treatment of malignant brain tumors. Our results are summarized as follows: (i) a recombinant HSV (M012) was constructed in which both copies of the γ134.5 gene were replaced with the bacterial CD gene, under the control of the cellular promoter Egr-1; (ii) M012-infected cells in vitro efficiently convert 5-fluorocytosine (5-FC) to 5-fluorouracil, thereby enhancing cytotoxicity of neighboring, uninfected cells; (iii) both direct and bystander cytotoxicity of murine neuroblastoma and human glioma cell lines after infection with M012 were demonstrated; (iv) direct intracerebral inoculation of A/J mice demonstrated lack of neurotoxicity at doses similar to G207, a γ134.5-deleted HSV with demonstrated safety in human patient trials and (v) intratumoral injection of M012 into Neuro-2a flank tumors in combination with 5-FC administration significantly reduced tumor growth versus tumors treated with R3659 combined with 5-FC, or treated with M012 alone. Thus, M012 is a promising new oncolytic HSV vector with an enhanced prodrug-mediated, antineoplastic effect that is safe for intracranial administration.

Preclinical Evaluation of a Genetically Engineered Herpes Simplex Virus Expressing Interleukin-12

ABSTRACT Herpes simplex virus 1 (HSV-1) mutants that lack the γ134.5 gene are unable to replicate in the central nervous system but maintain replication competence in dividing cell populations, such as those found in brain tumors. We have previously demonstrated that a γ134.5-deleted HSV-1 expressing murine interleukin-12 (IL-12; M002) prolonged survival of immunocompetent mice in intracranial models of brain tumors. We hypothesized that M002 would be suitable for use in clinical trials for patients with malignant glioma. To test this hypothesis, we (i) compared the efficacy of M002 to three other HSV-1 mutants, R3659, R8306, and G207, in murine models of brain tumors, (ii) examined the safety and biodistribution of M002 in the HSV-1-sensitive primate Aotus nancymae following intracerebral inoculation, and (iii) determined whether murine IL-12 produced by M002 was capable of activating primate lymphocytes. Results are summarized as follows: (i) M002 demonstrated superior antitumor activity in two different murine brain tumor models compared to three other genetically engineered HSV-1 mutants; (ii) no significant clinical or magnetic resonance imaging evidence of toxicity was observed following direct inoculation of M002 into the right frontal lobes of A. nancymae; (iii) there was no histopathologic evidence of disease in A. nancymae 1 month or 5.5 years following direct inoculation; and (iv) murine IL-12 produced by M002 activates A. nancymae lymphocytes in vitro. We conclude that the safety and preclinical efficacy of M002 warrants the advancement of a Δγ134.5 virus expressing IL-12 to phase I clinical trials for patients with recurrent malignant glioma.

Enhanced antiglioma activity of chimeric HCMV/HSV-1 oncolytic viruses.

Oncolytic herpes simplex virus (HSV)-1 γ134.5-deletion mutants (Δγ134.5 HSV) are promising agents for tumor therapy. The attenuating mutation renders the virus aneurovirulent but also limits late viral protein synthesis and efficient replication in many tumors. We tested whether one function of γ134.5 gene, which mediates late viral protein synthesis through host protein kinase R (PKR) antiviral response evasion, could be restored, without restoring the neurovirulence. We have previously reported the construction of two chimeric Δγ134.5 HSV vectors (chimeric HSV), C130 and C134, which express the human cytomegalovirus (HCMV) PKR-evasion genes TRS1 and IRS1, respectively. We now demonstrate the following. The HCMV/HSV-1 chimeric viruses (i) maintain late viral protein synthesis in the human malignant glioma cells tested (D54-MG, U87-MG and U251-MG); (ii) replicate to higher titers than Δγ134.5 HSV in malignant glioma cells in vitro and in vivo; (iii) are aneurovirulent; and (iv) are superior to other Δγ134.5 HSV with both improved reduction of tumor volumes in vivo, and improved survival in two experimental murine brain tumor models. These findings demonstrate that transfer of HCMV IRS1 or TRS1 gene into Δγ134.5 HSV significantly improves replication in malignant gliomas without restoring wild-type neurovirulence, resulting in enhanced tumor reduction and prolonged survival.

Enhanced inhibition of syngeneic murine tumors by combinatorial therapy with genetically engineered HSV-1 expressing CCL2 and IL-12

Herpes simplex viruses type 1 (HSV-1) that lack the γ134.5 gene are unable to replicate in the central nervous system (CNS), but maintain replication competence in actively dividing tumors. To determine if antitumor therapy by M002, a γ134.5− HSV that expresses interleukin-12 (IL-12), could be augmented by combinatorial therapy with another γ134.5-deleted HSV-1 engineered to express the chemokine CCL2, Neuro-2a tumors were established subcutaneously in the syngeneic A/J mouse strain. Tumors received multiple injections intratumorally either of saline, the parent, non-cytokine-expressing virus R3659, M002, M010 (γ134.5− HSV expressing CCL2), or a combination of M002 and M010. Efficacies were evaluated by monitoring inhibition of tumor growth over time. Results demonstrated the following: (1) inhibition of tumor growth was most pronounced in tumors treated with a combination of M002 and M010; (2) enhanced tumor growth inhibition for the combinatorial treatment group was statistically significant compared to either M002 or M010 alone; and (3) the variability between slopes of the tumor growth rates within an individual treatment group appeared to be virus-dependent, and was reproducible between experiments. Our results demonstrate that combinatorial cytokine/chemokine γ134.5− HSV therapies can provide superior antitumor effects in experimental tumors as a model for malignancies arising in the brain.

Recurrent Antiviral-Resistant Genital Herpes in an Immunocompetent Patient

Herpes simplex virus type 2 (HSV-2) resistance to antiviral drugs has been described primarily in immunocompromised patients. We report an apparently immunocompetent, human immunodeficiency virus-negative male patient who has experienced repeated HSV-2 genital outbreaks despite receiving antiviral prophylaxis with several different drugs. Several of the HSV-2 genital isolates from this patient have been confirmed as resistant to acyclovir and penciclovir. Antiviral resistance occurred in the setting of long-term prednisone treatment and intermittent acyclovir prophylaxis at suboptimal doses and persisted despite the cessation of oral steroid treatment. The patients genital herpes outbreaks were not controlled by high-dose prophylaxis with acyclovir, valacyclovir, and famciclovir. Cessation of antiviral prophylaxis resulted in reversion of this patients HSV-2 isolates to acyclovir and penciclovir sensitivity, although resistant virus reappeared when antiviral prophylaxis was resumed. Transmission of a sensitive HSV-2 strain from this patient to a female sex partner was observed. These observations confirm previous reports that resistance to acyclovir may develop during prophylactic therapy in an otherwise well, immunocompetent patient. These findings support the conclusion that both drug-sensitive and drug-resistant HSV-2 strains established latency in this patient and that both strains are capable of frequent reactivation.