Transcriptomic analysis of the innate immune response to in vitro transfection of plasmid DNA

Eric Warga; Jared Anderson; Matthew Tucker; Emily Harris; Jacob Elmer

doi:10.1016/j.omtn.2022.11.025

Transcriptomic analysis of the innate immune response to in vitro transfection of plasmid DNA

Mol Ther Nucleic Acids. 2022 Dec 5:31:43-56. doi: 10.1016/j.omtn.2022.11.025. eCollection 2023 Mar 14.

Authors

Eric Warga¹, Jared Anderson¹, Matthew Tucker¹, Emily Harris¹, Jacob Elmer¹

Affiliation

¹ Department of Chemical and Biological Engineering, Villanova University, White Hall, Room 119, 800 Lancaster Avenue, Villanova, PA 19085, USA.

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 identify genes that may influence transfection efficiency in four different cell types (PC-3, Jurkat, HEK-293T, and primary T cells). The highest transfection efficiency was observed in HEK-293T cells, which upregulated only 142 genes with no known antiviral functions after transfection with lipofectamine. Lipofection upregulated 1,057 cytokine-stimulated genes (CSGs) in PC-3 cells, which exhibited a significantly lower transfection efficiency. However, when PC-3 cells were transfected in serum-containing media or electroporated, the observed transfection efficiencies were significantly higher while the expression levels of cytokines and CSGs decreased. In contrast, lipofection of Jurkat and primary T cells only upregulated a few genes, but several of the antiviral CSGs that were absent in HEK-293T cells and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain the relatively low Lipofection efficiencies observed with T cells (8%-21% GFP⁺). Indeed, overexpression of one CSG (IFI16) significantly decreased transfection efficiency in HEK-293T cells.

Keywords: IFI16; IKBKE; MT: Oligonucleotides: Therapies and applications; MYD88; STING; cytokine-stimulated genes; cytokines; innate immune response; plasmid.