Transcriptomic Analysis of the Innate Immune Response toin vitroTransfection of Plasmid DNA

Abstract

The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA-sequencing to reveal correlations between the transfection efficiencies of four cell types (PC-3, Jurkat, HEK-293T, and primary CD3+ T cells) and their innate immune responses to nonviral gene delivery. Overall, the highest transfection efficiency was observed in HEK-293T cells (87%), which upregulated only 142 genes with no known anti-viral functions. Lipofection upregulated a much larger number (n = 1,057) of cytokine-stimulated genes (CSGs) in PC-3 cells, which also exhibited a significantly lower transfection efficiency. However, the addition of serum during Lipofection and electroporation significantly increased transfection efficiencies and decreased the number of upregulated genes in PC-3 cells. Finally, while Lipofection of Jurkat and Primary T cells only upregulated a few genes, several anti-viral CSGs that were absent in HEK and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain their relatively low Lipofection efficiencies (8-21%). Indeed, overexpression of one such CSG (IFI16) significantly decreased transfection efficiency in HEK cells to 33%.

Investigating dynamics of lentiviral vector secretion from HEK293T producer cells using a fractionated perfusion system

“…We wanted to test whether an intracellular immune response in HEK cells is activated by transient transfection similarly to activation of NFκB through TNF-α stimulation. [29,30] We also included groups stimulated with TNF-α at 3 μg mL −1 prior to being seeded (Figure 3A,B) to allow us to test whether TNF-α-induced inflammation impacts LV production dynamics. Titer was plotted alongside GLuc readout for each group to assess the effects of immune activation on LV production.…”