IN-VIVO L-DOPA PRODUCTION BY GENETICALLY-MODIFIED PRIMARY RAT FIBROBLAST OR 9L GLIOSARCOMA CELL GRAFTS VIA COEXPRESSION OF GTPCYCLOHYDROLASE-I WITH TYROSINE-HYDROXYLASE

To investigate the biochemical requirements for in vivo L-DOPA product ion by cells genetically modified ex vivo in a rat model of Parkinson' s disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer derma l fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway f or synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in th e absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6 -OHDA)-lesioned rats are minimal, the effects of cotransduction with h TH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L ce lls or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialy sis experiments showed that those subjects that received cells cotrans duced with hTH2 and hGTPCHI produced significantly higher levels of L- DOPA than animals that received either hTH2 or untransduced cells. How ever, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 week s after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD. (C) 1998 Academic Press.