Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro

Autologous bone marrow stromal cells engineered to produce 3.4-dihydroxyphe nylalanine(L-DOPA) can potentially be used as donor cells for neural transp lantation in Parkinson's disease. Here. we examined the possibility of usin g several different promoters and either a self-inactivating retrovirus (pS IR) or standard retroviruses to introduce into marrow stromal cells (MSCs), the two genes necessary for the cells to synthesize L-DOPA. pSIR vectors w ere constructed using the mouse phosphoglycerate kinase-1 (PGK) promoter or the cytomegalovirus (CMV) promoter to drive expression of either a GFP rep orter gene or a bicistronic sequence containing the genes for human tyrosin e hydroxylase type I (TH) and rat GTP cyclohydrolase I (GC) separated by an internal ribosome entry site (IRES). rMSCs were successfully transduced wi th both standard retroviral vectors and pSIR containing the PGK promoter. T ransduced rMSCs expressed GFP (90.4-94.4% of cells) or were able to synthes ize and secrete L-DOPA (89.0-283 pmols/10(6) cells/h). After transduced rMS Cs were plated at low density (3-6 cells/cm(2)), the cells expanded over 10 00-fold in 3-4 weeks, and the rMSCs continued to either express GFP or prod uce L-DOPA. Furthermore, two high-expressing clones were isolated and expan ded at low-density from rMSCs transduced with pSIR driven by the PGK promot er (97.0% GFP+ or 1096.0 pmols L-DOPA/10(6) cells/h).