Generation of DOPA-producing astrocytes by retroviral transduction of the human tyrosine hydroxylase gene: in vitro characterization and in vivo effects in the rat Parkinson model.
Astrocytes secreting high levels of L-3,4-dihydroxyphenylalanine (DOPA) have been generated by retrovirus-mediated transfer of the human tyrosine hydroxylase (TH) gene. Immature astrocytes obtained from prenatal rat brain were cocultured with TH virus producing psi-2 cells that had been pretreated with the mitosis inhibitor mitomycin-C. During the first week of coculture DOPA production gradually increased to reach a plateau after 7-9 days. At this time point virtually all cells were GFAP positive and over 80% of them expressed TH. DOPA production in the transduced astrocytes was largely independent of exogenous cofactor, and DOPA release into the medium was not influenced by addition of either KCl or tetrodotoxin or by removal of Ca2+ from the culture medium, indicating that the newly synthesized DOPA was constitutively released from the cells. Transplantation of the TH-transduced astrocytes to the striatum in unilaterally 6-hydroxydopamine lesioned rats reduced apomorphine-induced turning by about 50% at 2 weeks postgrafting. Microscopic analysis revealed that the transduced astrocytes survived very well after transplantation and that some of the grafted cells had migrated out, partly along blood vessels, into the surrounding striatum. TH expression was observed in cells with both the appearance of mature GFAP-positive astrocytes, as well as in more immature-looking cells. However, only a few percent of all transplanted cells maintained significant expression of the transgene, as determined by TH immuno-histochemistry. The results show that primary astrocytes may be highly useful as gene carriers for ex vivo gene therapy in the CNS. With future improvement in the gene transduction procedure for more efficient, sustained expression of the TH transgene in vivo, genetically engineered DOPA-producing astrocytes hold great promise as a tool to explore the potential of ex vivo gene therapy in Parkinson's disease.
Lundberg
C
C
Department of Medical Cell Research, Lund University, Sweden.
Horellou
P
P
Mallet
J
J
Björklund
A
A
D000818:Animals; D001253:Astrocytes / Q000201:enzymology / Q000378:metabolism* / Q000637:transplantation; D002455:Cell Division / Q000502:physiology; D017690:Cell Transplantation; D002478:Cells, Cultured / Q000166:cytology / Q000201:enzymology / Q000821:virology; D003587:Cytomegalovirus / Q000235:genetics; D004295:Dihydroxyphenylalanine / Q000096:biosynthesis* / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D017072:Neostriatum / Q000601:surgery; D010300:Parkinson Disease / Q000628:therapy*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D012190:Retroviridae / Q000235:genetics*; D019076:Transgenes / Q000502:physiology; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism
D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase