Efficient protection from methotrexate toxicity and selection of transduced human hematopoietic cells following gene transfer of dihydrofolate reductase mutants.
OBJECTIVE
While retrovirally mediated gene transfer of dihydrofolate reductase mutants (mutDHFR) has convincingly been demonstrated to confer methotrexate (MTX) resistance to murine hematopoietic cells, clinical application of this technology will require high efficacy in human cells. Therefore, we investigated retroviral constructs expressing various point mutants of human DHFR for their ability to confer MTX resistance to human clonogenic progenitor cells (CFU-C) and to allow for in vitro selection of transduced CFU-C.


METHODS
Primary human hematopoietic cells were retrovirally transduced using MMLV- and SFFV/MESV-based vectors expressing DHFR(Ser31), DHFR(Phe22/Ser31), or DHFR(Tyr22/Gly31). MTX resistance of unselected and in vitro-selected CFU-C was determined using MTX-supplemented methylcellulose cultures and gene transfer efficiency was assesed by single-colony PCR analysis.


RESULTS
While less than 1% mock-transduced CFU-C survived the presence of > or =5 x 10(-8) M MTX, MMLV- and SFFV/MESV-based vectors expressing DHFR(Ser31) significantly protected CFU-C from MTX at doses ranging from 2.5 to 30 x 10(-8) M. Vectors expressing DHFR(Phe22/Ser31) or DHFR(Tyr22/Gly31) were even more protective and MTX-resistant CFU-C were observed up to 1 x 10(-5) M MTX. Three-day suspension cultures in the presence of 10-20 x 10(-8) M MTX resulted in significant selection of mutDHFR-transduced CFU-C. The percentage of CFU-C resistant to 10 x 10(-8) M MTX increased fourfold to 20-fold and provirus-containing CFU-C increased from 27% to 79-100%.


CONCLUSIONS
Gene transfer of DHFR using suitable retroviral backbones and DHFR mutants significantly increases MTX resistance of human CFU-C and allows efficient in vitro selection of transduced cells using a short-term selection procedure.
Meisel
Roland
R
Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen, Hufelandstrasse 55, D-45122 Essen, Germany.
Bardenheuer
Walter
W
Strehblow
Claudia
C
Sorg
Ursula Regina
UR
Elmaagacli
Ahmet
A
Seeber
Siegfried
S
Flasshove
Michael
M
Moritz
Thomas
T
D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D017354:Point Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000494:pharmacology; D014161:Transduction, Genetic / Q000379:methods* / Q000592:standards
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate