Xandra O. Breakefield

Gene Therapy for Neurological Disorders and Brain Tumors

Part i: Vectors and Promoters. Introduction, J. Glorioso. Retrovirus Vectors and Regulatable Promoters, S. Reeves. Adenovirus Vectors, M. Cotten. Herpes Simplex Virus: Recombinant Vectors, P. A. Johnson. Herpes Simplex Virus: Amplicon Vectors, C. Fraefel, X. O. Breakefield, and D. Jacoby. Adeno-Associated Virus Vectors, F. I. Smith and T. J. McCown. Epstein-Barr Virus Vectors, J.-M. H. Vos, K. B. Quattrocchi, and B. J. Wendelburg. Lentiviral Vectors, D. Trono, U. Blomer, and L. Naldini. Promoters for Expression of Gene Products Within Neurons and Glia, J. W. Henson. Immunological Issues, J. G. Smith and S. L. Eck. Part ii: Neuro-oncology. Current Treatment Modalities for Brain Tumor, S. B. Tatter and G. R. Harsh IV. Experimental and Clinical Gene Therapies for Brain Tumors, E. A. Chiocca. Tumor Suppressor Gene Therapy for Brain Tumors, C. Gomez-Manzano, J. Fueyo, A. P. Kyritsis, W.K. A. Yung. Cytokine-Based Gene Therapy for Brain Tumors, J. H. Sampson, D. D. Bigner, and G. Dranoff. Delivery of Therapeutic Genes to Brain and Intracerebral Tumors, L. L. Muldoon, R. A. Kroll, M. A. Pagel, S. Roman-Goldstein, and E. A. Neuwelt. Rat Brain Tumor Models and Statistical Evaluation of Survival Data in Experimental Neuro-Oncology, R. F. Barth and M. L. Moeschberger. Part iii: Neurological Disorders. Neurological Disorders: An Overview, A. B. Young. Gene Transfer for Adult CNS Regeneration and Aging, M.C. Senut, I. Aubert, P.J. Horner, and F. H. Gage. Gene Therapy for Parkinsons Disease, M. C. Bohn and D. L. Choi-Lundberg. Gene Therapy for Ischemic Stroke, P. A. Pechan, M. Fujii, C. Fraefel, Andreas Jacobs, and M. A. Moskowitz. Gene Therapy for the Lysomal Storage Diseases, E. M. Kaye. Gene Therapy for Pain, G. Davar. Gene Therapy of Huntingtons Disease, O. Isaesen and N. Haque. Index.

Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150u2009kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal β-galactosidase (BGAL) gene of 100u2009kb. Temporary expression and functional activity of β-galactosidase (β-gal) could be detected in human β-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of β-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated β-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed β-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and β-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.

Virus-Mediated Genetic Treatment of Rodent Gliomas

The most common primary central nervous system neoplasm in adults is the malignant glioma. Approximately 5,000 new cases are diagnosed annually in the United States. This tumor has proven to be extremely refractory to currently available therapeutic modalities. A combination of aggressive surgical excision, radiation therapy, and chemotherapy has increased the life expectancy of patients suffering from this illness by only a few months (Schoenberg, 1983; Salcman, 1985; Kornblith et al., 1985). Sometimes the aggressive pursuit of these therapeutic modalities results in considerable neurologic dysfunction.

CHAPTER 3 – HSV Amplicon Vectors for Gene Delivery to the Nervous System

The HSV amplicon vector incorporates features of HSV-1, including a 150 kb transgene capacity, a viral origin of DNA replication and packaging signal, and virion proteins. The large transgene capacity is one of the most distinguishing features of this vector system, which allows incorporation of multiple transgenes and large genomic fragments, as well as informational elements from other virus vectors, including AAV, EBV, and retrovirus. Vectors have been modified to include elements, which increase infection of specific cell types and allow retention of transgene sequences, either as replicating episomal elements or through site-specific integration into the cell genome, and provide the ability to control transgene expression. The virion itself includes proteins that can be used to track infection and deliver fusion proteins to cells. Within the nervous system, these vectors are especially useful due to the natural neurotropism of the virus, with a strong retrograde component, and minimal perturbation of neuronal physiology. Vectors have been used to deliver proteins to facilitate fluorescence, bioluminescent, and magnetic resonance imaging, as well as to monitor neuronal functions in animal models involving learning/memory and addiction paradigms. Vectors have been designed to ameliorate symptoms in models of neurologic disease, including protection of neurons from toxic insults and replacement of genetically deficient proteins, as well as in treatment of brain tumors. Amplicon vectors are considered highly compatible with clinical trials due to their intrinsic lack of toxicity, but methods of production need to be improved to generate high titers and clinically compatible vector stocks.