The pathogeneses of the neurodegenerative disorders of aging, including Parkinson's disease (PD) remain poorly understood. In PD, neurotransmitter replacement strategy usually produces good symptomatic relief at least in the short term. Unfortunately, long-term systemic administration of the dopamine precursor, L-DOPA is often complicated by severe side effects. Therefore, methods that provide continuous localized delivery of catecholamine have been explored in recent years, with much emphasis on the employment of neural transplants. Genetically modified host cells provide potentially ideal autologous donor cells for neural transplantation since customized transgenes and gene products can be delivered directly to the striatum. The system also provides rigorous experimental conditions so that the in vivo effect of a specific transgene can be studied. We will attempt to regulate catecholamine synthesis by studying the roles of precursors and cofactors in catecholamine production in genetically modified cells. Primary cell cultures of fibroblasts will be co- transduced with genes for the neurotransmitter synthesizing enzyme (tyrosine hydroxylase [TH]) and the cofactor synthesizing enzyme (GTP cyclohydrolase I) in an attempt to enhance catecholamine production. Transduction will be done using recombinant retroviral vectors containing these genes. Recombinant retroviral vectors will be either dicistronic or separate vectors with distinguishable selection markers. Transduced cells will be characterized biochemically before grafting. The effect of grafting will be monitored biochemically by in vivo microdialysis and HPLC analysis, and functionally by apomorphine-induced rotations in rats with unilateral 6-hydroxydopamine lesions. Other behavioral tests not involving dopamine agonist stimulation will also be studied. Finally, TH and cofactor-producing cells will be co-grafted with cells transduced with aromatic L-amino acid decarboxylase cDNA so as to reconstitute the complete dopamine synthetic machinery in the grafts.
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