Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Michael A. Whitt, Ph.D.

Committee

Suzanne J. Baker, Ph.D. Lawrence M. Pfeffer, Ph.D. Tayebeh Pourmotabbed, Ph.D. Charles J. Russell, Ph.D.

Keywords

Glioblastoma Multiforme, Interferon, Matrix Protein, Oncolytic Virus, Vesicular Stomatitis Virus.

Abstract

Background: Over the past 30 years, little has changed in the treatment modalities and prognosis of patients suffering from Glioblastoma multiforme (GBM), the most common and by far the most devastating adult primary malignant brain tumor. Conventional therapies provide only a marginal increase in survival of GBM patients, post-diagnosis. Therefore, more novel means of treating GBM are needed to increase long-term survival and quality of life for those affected. Replication competent oncolytic viruses (OVs) have recently emerged as a possible option for treatment of high-grade gliomas. Particularly, recombinant vesicular stomatitis virus (rVSV), an enveloped, negative strand RNA virus, has shown promising results in preclinical studies. Tumor selectivity of VSV is thought to be associated with tumor specific defects in the interferon (IFN) pathway. However, largely due to insufficient attention on the role the immune system plays in efficacy of treatment, potential OVs have been obstructed from moving through the clinical trial pipeline past Phase I/II studies. rNCP12.1 is a novel recombinant VSV vector possessing specific mutations in the matrix protein. Thes mutations have been shown to promote viral attenuation in normal cells while maintaining cytotoxicity in a number of tumor cell lines. We aim to characterize and further develop this novel agent for the treatment of GBM. Methods: In order to determine differences between rNCP12.1 and wtVSV and to determine specificity of rNCP12.1 for tumor over normal cells, cell rounding assays, one-step growth curves, and cytotoxicity assays were performed in normal glial and tumor glial cell lines. To understand the basis of this selectivity and whether it correlated with the antiviral responses of IFN, expression levels of IFN and IFN stimulated genes (ISGs) were quantified, production of active IFN was measured, and the ability of cells to inhibit viral infection in response to exogenous IFN was determined. In vivo experiments were designed and carried out to test for oncolytic activity of rNCP12.1 in immunocompetent animal models of intracranial glioma. A single injection of rNCP12.1 was administered into previously implanted F98-GFP tumors. Tumor load and parameters of morbidity were assessed at 15 days following tumor implantation and long term at the time of euthanasia. Viral induced immune responses were assessed by the IFN bioassay and detection of circulating anti-VSV antibodies were achieved by Western blot analysis and a neutralizing antibody assay. Experimental methods of virus administration for treatment of glioma were further tested including multiple injections, injections using different VSV serotypes, continuous infusion of virus using implantable osmotic pumps, and pre-infected autologous carrier cells. These methods were designed to enhance anti-tumor effect by managing the negative effects of the tumor microenvironment and of a functional immune system on viral therapy. Results and Conclusion: rNCP12.1 was shown to be an attenuated strain of VSV that has clear differences in its growth and induction of the IFN response pathway in normal cells. It has a preference for growth in tumor cells as determined by viral titers, cell rounding, and cell viability post infection. This preference varied based on cellular expression of a particular IFN phenotype. The importance of this molecular versus histological cell profile was evident even in the performance of rNCP12.1 on human glioma cell lines that differ in their expression of IFN. In vivo evaluation of rNCP12.1 against a highly IFN resistant rat glioma cell line, F98, demonstrated its ability to decrease tumor size while eliciting a peripheral response to virus that protects normal tissue but also shortens its therapeutic window and the ability to sustain reduction of tumor over time. Several experimental methods in delivery of virus proved to be beneficial, including administering an additional dose of virus using a different serotype to bypass antiviral neutralizing responses and by shielding virus from the immune system through the use of tumor carrier cells. As an additional benefit, the latter was shown to have a unique pattern in eliciting tumor specific antibodies that was different from those increased by therapy with virus alone. This method also increased recovery of virus from brain tissue even after 20 days post treatment.

Our data supports the capability of rVSV vectors as treatment for GBM. Specifically rNCP12.1 therapy increased survival while decreasing tumor load, depending on method of administration. When given alone, virus is ultimately immunogenic and prompts anti-viral as well as anti-tumor immune responses. However, when shielded from the immune system, antiviral responses are minimal while anti-tumor responses are sustained. To this end, therapy cannot be fully addressed without addressing the effects the immune system has on therapy and on the host. Future studies, should include not only evaluation of tumor load, morbidity, and side effects of viral therapy but also immune responses especially those that are likely to enhance therapy past the acute stages of disease.

DOI

10.21007/etd.cghs.2013.0070

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