Protection of mice from methotrexate toxicity by ex vivo transduction using lentivirus vectors expressing drug-resistant dihydrofolate reductase.
Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.
Gori
Jennifer L
JL
Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church St. SE, University of Minnesota, Minneapolis, MN 55455, USA.
Podetz-Pedersen
Kelly
K
Swanson
Debra
D
Karlen
Andrea D
AD
Gunther
Roland
R
Somia
Nikunj V
NV
McIvor
R Scott
RS
D000818:Animals; D000931:Antidotes / Q000008:administration & dosage*; D001855:Bone Marrow Diseases / Q000139:chemically induced; D016026:Bone Marrow Transplantation; D004351:Drug Resistance / Q000235:genetics*; D005822:Genetic Vectors; D006400:Hematocrit; D016086:Lentivirus; D008727:Methotrexate / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000008:administration & dosage* / Q000235:genetics / Q000494:pharmacology*; D014161:Transduction, Genetic
D000931:Antidotes; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate