Abstract
Currently, low levels of stable gene transfer into hematopoietic tissues of large animals and humans continues to limit the clinical application of gene therapy. One strategy for overcoming this problem is to selectively expand, in vivo, the population of successfully gene-modified cells. Recent work has shown that nucleoside transport inhibition in combination with antifolates can be used to select in vivo for hematopoietic stem cells expressing drug-resistant dihydrofolate reductase (DHFR). In this study we investigated whether trimetrexate (TMTX) and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine ribose phosphate (NBMPR-P) can be used to select for tyr22-variant DHFR expressing transgenic hematopoietic cells in a murine bone marrow transplant model. Our results indicate that 40 mg/kg TMTX and 20 mg/kg NBMPR-P can be used in combination to expand transgene-positive progenitor cells 3- to 4-fold immediately following drug administration. In addition, long-term progenitor populations were expanded 2- to 3-fold in primary recipients, to approximately 5 months following drug administration. Secondary transplants conducted with marrow from primary recipients 5 months following drug administration revealed a statistically significant selective expansion of transgene-positive cells in the spleens and peripheral blood of these animals. No such expansion was observed in groups of mice treated with TMTX alone or NBMPR-P alone. We conclude that TMTX + NBMPR-P can be used to selectively expand transgenic tyr22-variant DHFR expressing murine hematopoietic stem cells in vivo.
Footnotes
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This work was supported by National Institutes of Health Minority Predoctoral Fellowship Award CA63916 to C.A.W. and Research Grant CA60803 to R.S.M.
Abbreviations
- MDR-1
- multidrug resistance gene
- DHFR
- dihydrofolate reductase
- TMTX
- trimetrexate
- NBMPR-P
- nitrobenzylmercaptopurine ribose phosphate
- tyr22
- L22Y-substituted dihydrofolate reductase
- MTX
- methotrexate [(+)-amethopterin)]
- PBS
- phosphate-buffered saline
- Received June 1, 2001.
- Accepted September 27, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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