期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 112, 期 7, 页码 1429-1436出版社
WILEY
DOI: 10.1002/bit.25538
关键词
Sleeping Beauty transposon system; cell engineering; multiplexed gene delivery; immunotherapy
资金
- Washington Research Foundation
- Ben Towne Foundation
- National Science Foundation Graduate Research Fellowship [2013163401]
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 (approximate to 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. (c) 2015 Wiley Periodicals, Inc.
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