Engineering Human T Cells for Resistance to Methotrexate and Mycophenolate Mofetil as an In Vivo Cell Selection StrategyReport as inadecuate




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Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic-immunosuppressive drugs methotrexate MTX and mycophenolate mofetil MMF in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase DHFRFS; L22F, F31S and inosine monophosphate dehydrogenase II IMPDH2IY; T333I, S351Y conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically up to 0.1 µM MTX and 1.0 µM MPA. Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt+DHFRFS+IMPDH2IY+ T cells could be enriched following adoptive transfer either by systemic administration of MTX alone 4.4 -fold, MMF alone 2.9-fold, or combined MTX and MMF 4.9-fold. These findings demonstrate the utility of both DHFRFS-MTX and IMPDH2IY-MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.



Author: Mahesh Jonnalagadda, Christine E. Brown, Wen-Chung Chang, Julie R. Ostberg, Stephen J. Forman, Michael C. Jensen

Source: http://plos.srce.hr/



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