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

Gene Therapy volume 8, pages 1214–1223 (2001)Cite this article

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/106 cells/h). After transduced rMSCs were plated at low density (3–6 cells/cm2), 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/106 cells/h).

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Acknowledgements

We thank E Javazon for critical review of the manuscript. This work was supported in part by NIH grants AR47796 and AR44210, a gift from the Oberkotter Foundation and The Louisiana Gene Therapy Research Consortium.

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Authors and Affiliations

  1. Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA, USA

    EJ Schwarz, RL Reger & DJ Prockop

  2. Department of Neurology, MCP Hahnemann University, Philadelphia, PA, USA

    GM Alexander & SA Azizi

  3. Department of Radiation Oncology, MCP Hahnemann University, Philadelphia, PA, USA

    R Class

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Schwarz, E., Reger, R., Alexander, G. et al. Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro. Gene Ther 8, 1214–1223 (2001). https://doi.org/10.1038/sj.gt.3301517

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