Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity

Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body i rradiation (TBI), To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI wou ld allow sufficient engraftment of MTX-resistant hematopoietic cells to con fer survival on recipient mice administered MTX, FVB/N animals were adminis tered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 10(7) FVB/ N transgenic marrow cells expressing an MTX-resistant dihydrofolate reducta se (DHFR) transgene, and then administered MTX daily for 60 days, Control m ice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all a nimals transplanted with transgenic marrow survived MTX administration, reg ardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of T BI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subseque ntly administered MTX when compared with control animals administered phosp hate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 x 10(6) and 1 x 10(6) cells) of DHFR transgenic donor marrow, resulting in low-level (appr oximately 1%) engraftment, In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hemato poietic stem cells,(Blood, 2000;96:1334-1341) (C) 2000 by The American Soci ety of Hematology.