Multiplexed gene transfer to a human T-cell line by combining Sleeping Beauty transposon system with methotrexate selection.

Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance T-cell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, non-viral gene transfer to a human T-cell line with efficient selection (∼ 50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade T-cells housing multiplexed genetic modifications.