The overall goal of the proposed research is to use animal models of neuropathic lysosomal storage disorders (LSD) to develop and evaluate novel ex vivo and in vivo gene transfer strategies for the treatment of disorders whose primary site of pathology is in the central nervous system (CNS). To facilitate these studies we have established breeding colonies of cats with mucopolysaccharidosis Type I and GM2 gangliosidosis and constructed 'knock-out' mouse model of Schindler disease. Central to this application is the fact that, unlike other disorders causing mental retardation in which the primary metabolic defect lies outside the CNS, the primary cell type which must be targeted in these lysosomal models are the neurons of the CNS. The focus, therefore, of our research will be to develop and evaluate in animal model systems methods to deliver genes to the CNS and to target them (and/or their gene products) to neurons for high-level expression. Four projects comprise this application. Project 1 is aimed at developing vectors which oxerexpress and secrete lysosomal enzymes that are targeted for neuronal uptake. For these studies, lysosomal cDNAs will be placed under the control of a strong universal promoter so that they can be introduced into any cell type and efficiently secrete the therapeutic protein. Project 2 will study the use of neural cell-type specific promoters to drive overexpression and secretion of lysosomal enzymes from specific neural cells in the CNS. Expression cassettes containing the lysosomal cDNAs will be developed containing the neurofilament subunit (neuron specific), myelin basic protein (oligodendrocyte specific) and glial fibrillary acid protein (astrocyte specific) promoter sequences and evaluated in transgenic mice to determine the levels of lysosomal enzyme expression, secretion and reuptake by neural cells. Optimal lysosomal fusion protein constructs developed in Project 1 will be evaluated using these expression cassettes and the efficacy of universal vs. cell-type specific promoters will be studied. Project 3 will evaluate the migration and persistance of neuroglial cells after implantation into the developing mammalian brain. Specifically, this project will evaluate the persistance and movement of oligodendrocyte, astrocyte and microglia after transplantation into CNS tissue and determine which neural cells are most appropriate for ex vivo transduction with the lysosomal overexpression vectors. Project 4 will use the murine and feline neuropathic LSD animal models to evaluate the biochemical, pathological and clinical efficacy of the various strategies developed in Projects 1-3 to deliver the lysosomal overexpression/secretion vectors to the CNS. These will include ex vivo delivery via hematopoietically-derived microglia and/or transplanted neural cells and in vivo delivery using virus vectors and/or carotoid infusion of expression vector/protein conjugates with and without transient disruption of the blood brain barrier. The four Projects will be supported within the Program by three Cores: Administrative (Core A), Laboratory Animal (Core B) and Microscopy (Core C).
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