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Multiplexed gene transfer to a human T‐cell line by combining Sleeping Beauty transposon system with methotrexate selection
Author(s) -
Kacherovsky Nataly,
Liu Gary W.,
Jensen Michael C.,
Pun Suzie H.
Publication year - 2015
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25538
Subject(s) - nucleofection , sleeping beauty transposon system , biology , cell culture , gene , transfection , microbiology and biotechnology , transposable element , mutant , genetics
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. Biotechnol. Bioeng. 2015;112: 1429–1436. © 2015 Wiley Periodicals, Inc.