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

Abstract

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 devices, cellular states, and materials to be delivered. Here, 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 human 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. Here, we outline the best practices for achieving highly-efficient 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 better anti-tumor activity for immunotherapy, manipulating naive T cells with high efficiency is also of high importance to clinical applications and to study the biology of these cells.

A tissue-like platform for studying engineered quiescent human T-cells’ interactions with dendritic cells

“…Three different settings were tested: V-24, U-14 and T-23. For Neon electroporation, 2.5 × 10 6 cells were resuspended in a 100 μl tip, and two settings were tested (1700 V 10 ms, 4 pulses and 2150 V, 20 ms one pulse) (Aksoy et al, 2019).…”

Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation

“…Physical delivery methods have recently gained attention when it comes to in vitro and ex vivo cell modification, featuring a broad applicability on different cell types and cargos [17,25]. Electroporation, which makes use of strong electric fields to deliver nucleic acids to the cell interior, is currently the preeminent tool for mRNA transfections of hard-to-transfect immune cells [2,26]. It should be noted, however, that electroporation was amply shown to come with significant loss of cell viability, induction of unwanted phenotypic changes or loss of cell functionality [17,[27][28][29][30].…”

Inhibition of Caspases Improves Non-Viral T Cell Receptor Editing

“…Despite these advantages, electrotransfer may result in severe cell death [ 12 , 13 ]. The viability of human primary T cells in gene electrotransfer experiments is highly dependent on the donors, varying from 20% to 40% under optimized experimental conditions for achieving adequate electrotransfer efficiency (e.g., 40%) [ 13 , 14 , 15 ]. The low viability is a major issue that needs to be tackled before the technology can be applied successfully to manufacturing CAR-T cells.…”