γ-Glutamylcysteine Synthetase and L-Buthionine-( S,R )-Sulfoximine: A New Selection Strategy for Gene-Transduced Neural and Hematopoietic Stem/Progenitor Cells

In most experimental gene therapy protocols involving stem/progenitor cells, only a small fraction of cells, often therapeutically inadequate, can be transduced and made to 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 explore the potential of the heavy subunit of γ-glutamylcysteine synthetase (γ-GCSh) to act as a selection marker. We found that 3T3 fibroblasts transduced with the bicistronic retroviral vector SF91/GCSh-eGFP, encoding γ-GCSh and the enhanced green fluorescent protein (eGFP), were highly resistant to L-buthionine-(S,R)-sulfoximine (BSO), a γ-GCS inhibitor with a 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 γ-GCS subunits had higher intracellular GSH levels, and a lower level of resistance to the cytotoxic activity of BSO, compared with cells overexpressing γ-GCSh alone. 3T3 fibroblasts overexpressingγ-GCSh could be selected from cultures containing both naive 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 (HSCs/HPCs) from mouse bone marrow, transduced with the γ-GCSh-eGFP vector, could be selected by BSO treatment in vitro. On ex vivo BSO selection and reimplantation into a syngeneic myeloablated host, donor HSCs/HPCs repopulated the marrow and continued to express the transgene(s). These results provide proof of principle that somatic stem/progenitor cells, transduced simultaneously with a potentially curative gene and γ-GCSh, can be selected by treatment with BSO before in vivo transplantation.