Gamma-glutamyl cysteine synthetase and L-buthionine-S,R-sulfoximine (BSO): a new selection strategy for gene-transduced neural and hematopoietic stem/progenitor cells.

Abstract

In most experimental gene therapy protocols involving stem/progenitor cells, only a small fraction of cells, often therapeutically inadequate, can be transduced and express the therapeutic gene. A promising strategy for overcoming this problem is the use of a dominant selection marker, such as a drug-resistance gene. In this paper, we have explored the potential of the heavy subunit of gamma-glutamyl cysteine synthetase (gamma-GCSh) to act as a selection marker. We found that 3T3 fibroblasts transduced with a bicistronic retroviral vector, coding for gamma-GCSh and the enhanced green fluorescent protein (eGFP), were highly resistant to L-buthionine-S,R-sulfoximine (BSO), a gamma-GCS inhibitor with a very low clinical toxicity profile. The level of resistance was not proportional to the increase in intracellular glutathione. In fact, cells overexpressing both heavy and light gamma-GCS subunits had higher intracellular GSH levels, and a lower level of resistance to the cytotoxic activity of BSO compared to cells overexpressing gamma-GCSh alone. 3T3 fibroblasts overexpressing gamma-GCSh could be selected from cultures containing both naïve and gene-modified cells by application of exogenous BSO selection pressure for 4 days. Also primary neural stem/progenitor cells derived from the lateral ventricles of mouse neonatal brains and primary hematopoietic stem/progenitor cells from mouse bone marrow, transduced with the gamma-GCSh-eGFP vector, could be selected by BSO treatment in vitro. These results provide proof-of-principle that neural and hematopoietic stem/progenitor cells, transduced with a potentially curative gene and co-expressing the simultaneously transduced gamma-GCSh gene, can be selected by treatment with BSO