title: Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro.
abstract: Autologous bone marrow stromal cells engineered to produce 3,4,-dihydroxyphenylalanine (L-DOPA) can potentially be used as donor cells for neural transplantation in Parkinson's disease. Here, we examined the possibility of using several different promoters and either a self-inactivating retrovirus (pSIR) or standard retroviruses to introduce into marrow stromal cells (MSCs), the two genes necessary for the cells to synthesize L-DOPA. pSIR vectors were constructed using the mouse phosphoglycerate kinase-1 (PGK) promoter or the cytomegalovirus (CMV) promoter to drive expression of either a GFP reporter gene or a bicistronic sequence containing the genes for human tyrosine hydroxylase type I (TH) and rat GTP cyclohydrolase I (GC) separated by an internal ribosome entry site (IRES). rMSCs were successfully transduced with both standard retroviral vectors and pSIR containing the PGK promoter. Transduced rMSCs expressed GFP (90.4--94.4% of cells) or were able to synthesize and secrete L-DOPA (89.0--283 pmols/10(6) cells/h). After transduced rMSCs were plated at low density (3--6 cells/cm(2)), the cells expanded over 1000-fold in 3--4 weeks, and the rMSCs continued to either express GFP or produce L-DOPA. Furthermore, two high-expressing clones were isolated and expanded at low-density from rMSCs transduced with pSIR driven by the PGK promoter (97.0% GFP+ or 1096.0 pmols L-DOPA/10(6) cells/h).
authors.affiliation: Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA 70112, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Schwarz
authors.forename: R L
authors.initials: RL
authors.lastname: Reger
authors.forename: G M
authors.initials: GM
authors.lastname: Alexander
authors.forename: R
authors.initials: R
authors.lastname: Class
authors.forename: S A
authors.initials: SA
authors.lastname: Azizi
authors.forename: D J
authors.initials: DJ
authors.lastname: Prockop
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism* / Q000821:virology*; D002455:Cell Division; D002478:Cells, Cultured; D002999:Clone Cells; D006136:GTP Cyclohydrolase / Q000235:genetics; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D007980:Levodopa / Q000096:biosynthesis*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D010300:Parkinson Disease / Q000628:therapy*; D011401:Promoter Regions, Genetic; D051381:Rats; D011917:Rats, Inbred Lew; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D007980:Levodopa; D014446:Tyrosine 3-Monooxygenase; D006136:GTP Cyclohydrolase