Abstract
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.
Highlights
A continuing unmet need for genetically engineered cellular therapies is the development of drug selection platforms that are non-immunogenic, and, that enable selection to occur either in vitro or in vivo in humans
Cells for MTX and mycophenolate mofetil (MMF) Resistance To compare MTX- and MMF-mediated cell selection strategies, either singly or in combination, we designed a lentiviral vector to direct the co-expression of DHFRFS, IMPDH2IY and a truncated human EGF receptor [22]
We chose to evaluate MTX and MMF drug selection in central memory derived T (TCM) cells, a subpopulation of CD62L+CD45RO+ T cells, which have been shown to have favorable properties for therapeutic application including the capacity for self renewal, proliferation, long-term persistence, and an ability to differentiate into effector T cells [10,15,24]
Summary
A continuing unmet need for genetically engineered cellular therapies is the development of drug selection platforms that are non-immunogenic, and, that enable selection to occur either in vitro or in vivo in humans. While a number of drug-resistance enzymes have been employed for selection of gene modified cells, including O6-mehtylguanine-DNA-methyltransferease (MGMT), multidrug resistance associated protein 1 (MDR1), bacterial hygromycin resistance gene (Hy) and neomycin phosphotransferase (neo) variants [1], many of these selective transgenes have proven disadvantageous in the clinic. Transgenes of non-human origin used for in vitro selection (e.g., Hy, neo, and Herpes simplex thymidine kinase, HSV-tk), often lead to immunological rejection of gene-modified cells [2,3,4,5,6]. There is a need for alternative strategies that will enable drug selection of gene modified cells with a tolerable toxicity profile in human patients
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