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Viable and efficient electroporation-based genetic manipulation of unstimulated human T cells

View ORCID ProfilePinar Aksoy, View ORCID ProfileBülent Arman Aksoy, View ORCID ProfileEric Czech, View ORCID ProfileJeff Hammerbacher
doi: https://doi.org/10.1101/466243
Pinar Aksoy
Microbiology and Immunology Department at the Medical University of South Carolina
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Bülent Arman Aksoy
Microbiology and Immunology Department at the Medical University of South Carolina
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Eric Czech
Microbiology and Immunology Department at the Medical University of South Carolina
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Jeff Hammerbacher
Microbiology and Immunology Department at the Medical University of South Carolina
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  • For correspondence: hammer@hammerlab.org
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Abstract

Genetic manipulation of human primary T cells is a valuable technique for basic research in immunology to explore gene function and for discovering novel clinical applications. Electroporation is the most feasible non-viral material delivery system for manipulating human T cells given its time- and cost-effectiveness. However, efficient delivery requires electroporation settings to be optimized for different electroporation devices, cellular states, and materials to be delivered. In this study, we used electroporation to either induce exogenous gene expression in human primary T cells by plasmids or in vitro transcribed (IVT) mRNA and also target endogenous genes by Cas9 ribonucleoproteins (RNPs). We characterized the electroporation conditions both for activated and unstimulated T cells. Although naive cells are non-dividing and therefore their genetic manipulation is harder compared to activated T cells, we developed the technical ability to manipulate both naive and memory cells within the unstimulated T cell population by IVT mRNA and Cas9 RNP electroporation with more than 95% and 80% efficiency, respectively, and by plasmids with more than 50% efficiency. Here, we outline the best practices for achieving highly-efficient and non-viral genetic manipulation in primary T cells without causing significant cytotoxicity to the cells. Because there is increasing evidence for “less-differentiated” T cells to have a better anti-tumor activity for immunotherapy, manipulating naive T cells with high efficiency is also of high importance to clinical applications. Furthermore, manipulation of naive T cells without the need for activation is important for studying the biology of these cells.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted April 13, 2019.
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Viable and efficient electroporation-based genetic manipulation of unstimulated human T cells
Pinar Aksoy, Bülent Arman Aksoy, Eric Czech, Jeff Hammerbacher
bioRxiv 466243; doi: https://doi.org/10.1101/466243
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Viable and efficient electroporation-based genetic manipulation of unstimulated human T cells
Pinar Aksoy, Bülent Arman Aksoy, Eric Czech, Jeff Hammerbacher
bioRxiv 466243; doi: https://doi.org/10.1101/466243

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