Protection of Mice from Methotrexate Toxicity by ex Vivo Transduction Using Lentivirus Vectors Expressing Drug-Resistant Dihydrofolate Reductase

Abstract

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. MTXtreated 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% vectormodified 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.

Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

“…Fortyeight hours later, DNA was extracted from the exposed cells and vector copy number was determined by quantitative PCR for the integrated lentiviral strong stop sequence or for the GFP sequence as previously described. 23 Concentrated vector titers ranged from 10 8 /ml to 10 9 /ml. CSIIEG was also titered for GFP expression by flow cytometry as previously described.…”

“…CSIIEG was also titered for GFP expression by flow cytometry as previously described. 23 Ex vivo lentiviral transduction and bone marrow transplantation All procedures were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee. A murine model of Artemis deficiency backcrossed onto C57BL/6 background and exhibiting no leakiness 20 was maintained under specific pathogen-free conditions at the University of Minnesota and further bred onto both CD45.1 and CD45.2 congenic backgrounds.…”

“…23,24 Briefly, 1.4 · 10 7 HEK 293T cells were seeded into poly-llysine-coated 15-cm 2 plates and cultured overnight in DMEM supplemented with 1% penicillin-streptomycin (Pen Strep) and 8% FBS. Lentiviral vector plasmid constructs were cotransfected with pDNRF, encoding lentiviral structural and enzymatic proteins, and pMD.G, encoding VSV-G envelope protein, and incubated at 37°C with 10% CO 2 .…”

“…Vector copy number was determined by quantitative PCR for the lentiviral strong stop sequence as described. 23 In vivo KLH challenge Test animals were immunized by intraperitoneal injection of 100 lg of NP-KLH (4-hydroxy-3-nitrophenylacetyl hapten-conjugated keyhole limpet hemocyanin) (Biosearch Technologies, Novato, CA) and boosted 5 weeks later. Before immunization and 1 week after the boost injection, sera were collected, aliquoted, and stored at -20°C for analysis by enzyme-linked immunosorbent assay (ELISA).…”

“…Luciferase expression plasmids pAPro-Luc (pGL3-APro 19 ) and pPGK-Luc (pGL3-based 22 ), and the simian virus 40 (SV40)-regulated plasmid pGL3-C (Clontech, Palo Alto, CA) have been previously described. Plasmid pEf1a-Luc was assembled by ligation of a 1102-bp XhoI fragment (including the EF1a promoter) isolated from pDL2G 23 into pGL3-Basic at the XhoI site upstream of the luciferase coding sequence.…”