773. Chemoprotection of Mouse Marrow Transplant Recipients by Ex Vivo Lentiviral Transduction of Murine tyr22 Dihydrofolate Reductase Conferring Resistance to Methotrexate
Authors: Jennifer L. Gori, Kelly Podetz-Pederson, Debra Swanson, Andrea D. Karlin, Nikunj V. Somia, R. Scott McIvor
Journal: Molecular Therapy (2006)

Top of pageAbstract
Methotrexate (MTX) impedes tumor development by inhibiting dihydrofolate reductase (DHFR), an enzyme that catalyzes the conversion of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate (THF). MTX depletes cells of THF, a required precursor for de novo purine and thymidine nucleotide synthesis, and has toxic effects on normal cells of the hematopoietic and gastrointestinal systems. To decrease MTX sensitivity, we introduced a MTX-resistant murine DHFR variant into mouse bone marrow by lentiviral transduction, so as to efficiently transduce non-dividing hematopoietic stem cells. We constructed HIV-1-based lentiviral vectors containing an enhanced green fluorescent protein (eGFP) gene with or without a murine tyr22 DHFR cDNA under transcriptional control of the human EF1-|[alpha]| promoter. These VSV-G-protein pseudotyped lentiviral vectors contained a self-inactivating 3|[prime]| LTR, rev response element (RRE) and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to enhance transduction efficiency. Viral vector was produced by co-transfection in 293T cells and concentrated 100-fold by centrifugation to a titer of 1 x 108 IU/ml, as determined by MTX-resistant colony formation and by flow cytometry for GFP expression. We transduced unfractionated bone marrow from normal Bl/6 Ly5.1 mice with either DHFR-GFP or GFP virus for 12 hours at a multiplicity of infection of 10 in the presence of cytokines (mSCF, IL-6, IL-3). Congenic Ly5.2 recipients received sub-lethal irradiation (700 cGy) prior to transplantation of 3 x 106 transduced marrow cells. MTX or PBS were administered daily starting twenty- four hours after transplantation in three recipient cohorts. Animals that received DHFR-virus transduced marrow recovered to normal hematocrit levels by week 3 in spite of MTX administration (44.7% +/- 2.5). In contrast, animals that received GFP-transduced marrow with subsequent MTX administration exhibited extremely reduced hematocrits (16.5% +/- 0.5) at week 3 and did not survive MTX dose-escalation to 2 mg/kg/day. Animals on MTX therapy that received DHFR-GFP transduced marrow exhibited increased donor cell (Ly5.1) engraftment and significant GFP marking in lymphoid (CD3, B220) and myeloid (GR-1) populations, while recipients that received GFP-transduced marrow exhibited lower levels of peripheral blood mononuclear cells and GFP+ leukocytes. Studies are currently underway to determine transgene copy number in repopulated marrow cells and assess MTX toxicity for the gastrointestinal tract. We conclude that transplantation of lentivirally transduced MTXr-DHFR transgenic marrow supports bone marrow repopulation during MTX chemotherapy in irradiated recipients, providing significant chemoprotection that permits MTX dose-escalation. These pre-clinical results support the development of a clinical gene therapy protocol to provide chemoprotection by lentiviral gene transfer.