Gamma-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 gamma-glutamylcysteine synthetase (gamma-GCSh) to act as a selection marker. We found that 3T3 fibroblasts transduced with the bicistronic retroviral vector SF91/GCSh-eGFP, encoding 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 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 with cells overexpressing gamma-GCSh alone. 3T3 fibroblasts overexpressing gamma-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 gamma-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 gamma-GCSh, can be selected by treatment with BSO before in vivo transplantation.
Lorico
Aurelio
A
Department of Tumor Biology, Norwegian Radium Hospital, Montebello, Oslo 0310, Norway. alorico@usouthal.edu
Bratbak
Daniel
D
Meyer
Johann
J
Kunke
David
D
Krauss
Stefan
S
Plott
Walter E
WE
Solodushko
Victor
V
Baum
Christopher
C
Fodstad
Oystein
O
Rappa
Germana
G
D016475:3T3 Cells / Q000187:drug effects; D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D002478:Cells, Cultured; D018432:Drug Resistance, Multiple; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D012641:Selection, Genetic; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D014161:Transduction, Genetic / Q000379:methods*
D049452:Green Fluorescent Proteins; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione