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A gene transfer strategy for making bone marrow cells resistant to
trimetrexate
HT Spencer, SE Sleep, JE Rehg, RL Blakley and BP Sorrentino
Department of Biochemistry, St Jude Children's Research Hospital, Memphis,
TN 38101 USA.
Trimetrexate (TMTX) is an anticancer drug with potential advantages over
the more commonly used antifolate, methotrexate (MTX); however, its use has
been limited by severe myelosuppression. Retroviral vectors containing
mutant dihydrofolate reductase (DHFR) genes have been used to protect bone
marrow cells from MTX, suggesting a similar approach could be used for
TMTX. We first screened six variants of human DHFR to determine which
allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing
a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a
100-fold increase in resistance over controls. Murine hematopoietic
progenitor cells transduced with an L22Y- containing retroviral vector also
showed high-level TMTX resistance in vitro. Mice reconstituted with
L22Y-transduced bone marrow cells were challenged with a 5-day course of
TMTX to determine whether hematopoiesis could be protected in vivo.
Transfer of the L22Y vector resulted in consistent protection from
TMTX-induced neutropenia and reticulocytopenia at levels that correlated
with the proviral copy number in circulating leukocytes. We conclude that
the L22Y vector is highly effective in protecting hematopoiesis from TMTX
toxicity and may provide a means for increasing the therapeutic utility of
TMTX in certain cancers.
Volume 87,
Issue 6,
pp. 2579-2587,
03/15/1996
Copyright © 1996 by The American Society of Hematology
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