REGULATION OF DOPAMINE PRODUCTION BY GENETICALLY-MODIFIED PRIMARY FIBROBLASTS

Primary skin fibroblasts were genetically modified with catecholamine-synthesizing enzyme genes and studied as potential syngeneic donor cells to supply catecholamines in animal models of Parkinson's disease. Primary skin fibroblasts obtained from inbred Fischer 344 rats were transduced with tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) cDNAs using retroviral vector system. The transduced cells were characterized in vitro by enzymatic assay, immunocytochemistry, and HPLC analysis of catecholamine production and release. Accumulation of high levels of dopamine was detected in the media in a time-dependent manner. Secretion of dopamine and its metabolites appeared to be constitutive without significant storage capacity in vesicles or regulation at the level of secretion. The feasibility of regulating the final dopamine production by the AADC-transduced cells was explored in two ways. First, administration of various doses of the precursor, L-dopa, resulted in a controlled production of dopamine by these cells. Second, coculturing AADC-transduced cells with TH-transduced cells in various proportions allowed control of dopamine production. TH-transduced cells served as an endogenous source of precursor. We propose the use of these cells to study the role of AADC in restoring the dopamine-deficient behavior and to compare the effect of dopamine-producing cells with L-dopa-producing cells either by co-grafting TH-transduced cells with AADC-transduced cells or by grafting TH-transduced cells alone. The role of AADC in vivo will be assessed in future experiments involving animal models of Parkinson's disease.