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 production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal 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 for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks 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.
Leff
S E
SE
Department of Gene Therapy Applications, Cell Genesys Inc., 342 Lakeside Drive, Foster City, California, 94404, USA. sleff@flash.net
Rendahl
K G
KG
Spratt
S K
SK
Kang
U J
UJ
Mandel
R J
RJ
D016475:3T3 Cells; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D000978:Antiparkinson Agents / Q000494:pharmacology; D001058:Apomorphine / Q000494:pharmacology; D001522:Behavior, Animal / Q000187:drug effects; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D003342:Corpus Striatum / Q000737:chemistry / Q000201:enzymology / Q000473:pathology; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000637:transplantation; D006136:GTP Cyclohydrolase / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015316:Genetic Therapy; D018316:Gliosarcoma; D006801:Humans; D007980:Levodopa / Q000096:biosynthesis*; D008297:Male; D051379:Mice; D017551:Microdialysis; D010302:Parkinson Disease, Secondary / Q000378:metabolism / Q000601:surgery / Q000628:therapy*; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012190:Retroviridae / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000637:transplantation; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D000975:Antioxidants; D000978:Antiparkinson Agents; D011993:Recombinant Fusion Proteins; D001708:Biopterins; D007980:Levodopa; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; D006136:GTP Cyclohydrolase; C003402:sapropterin; D001058:Apomorphine