This project is directed toward gene therapy for brain tumors by developing novel herpes simplex virus type 1 (HSV) derived vectors and evaluating their efficacy and toxicity in rodent models. The focus will be on: achieving gene delivery to tumor cells which have migrated away from the main mass; extending delivery to both dividing and non-dividing tumor cells; increasing the selectivity of therapeutic gene expression for tumor cells; and evaluating toxicity of the vectors. Recombinant virus and amplicon type vectors will be combined to achieve replication- conditional and replication-incompetent vector systems. Recombinant virus vectors will be combined to achieve replication-conditional and replication-incompetent vector systems. Recombinant virus vectors will serve as helper virus for the amplicon vector and will bear mutations in the immediate early gene, ICP27, which is essential for virus replication, as well as other virus gene, e.G. gamma 34.5, to reduce neurovirulence, and ribonucleotide reductase to make virus propagation selective for dividing cells. For replication-conditional vectors, the ICP27 gene will be placed in the amplicon under tumor-selective promoter elements, including the GFAP promoter, and the nestin enhancer. In this 'piggy back' system, propagation of the amplicon and recombinant virus vectors would be mutually dependent and cell-selective. Amplicons will be further modified so as to enhance their retention in dividing tumor cells by incorporation of elements from the adenoassociated virus (AAV)-, the rep gene and the ITRs, needed for integration into the host cell genome. Further, we will generate therapeutic vectors by incorporation of genes for prodrug-activating enzymes, cytochrome P4502B1 and HSV-thymidine kinase under control of tumor-selective regulatory elements. We will attempt to expand the range of the by-stander effect by targeting the prodrug activating enzyme to the cell surface so that toxic metabolites can be generated in the extracellular space. Gene delivery will be assessed in rat and human glioma lines and control lines in culture. Gene delivery to tumor cells and normal cells, as well as neuropathogenicity and systemic toxicity, will also be evaluated in rodents bearing intracerebral tumors following administration of the vectors directly into the tumor mass, through the ventricles or through the blood stream with temporary disruption of the blood-brain-barrier. Models will be developed to explore the effects of reactivation of latent wild type virus, immune response to the virus, and generation of recombinant virus. In combination with other projects in this Program these studies should provide a new generation of vectors to be evaluated in preclinical trials for potential use in humans.
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