Transcriptomic Analysis of the Innate Immune Response to Plasmid DNA in vitro

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 Lipofection in serum-free media (SFM). Overall, the highest transfection efficiency was observed in HEK-293T cells, 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 exhibited a significantly lower transfection efficiency. However, the addition of serum during Lipofection and electroporation provided much higher transfection efficiencies and lower number of upregulated genes. This inverse correlation between transfection efficiency and the number of differentially expressed genes was contradicted by the T cell lines that achieved lower transfection efficiencies and only upregulated a few upregulated genes following Lipofection. However, several of the CSGs that were absent in HEK and upregulated in PC-3 cells were constitutively expressed in T cells, suggesting that T cells constitutively express CSGs to prevent infection. 3 cell lines transfected with Lipofectamine in serum-free media (SFM), 6 samples per cell line with (3) untransfected and (3) transfected samples; plus 12 PC-3 samples that were either Lipofected or electroporated in SCM (30 samples overall)