Unlocking the Puzzle of Mammalian Transfection: The Role of the RNA-sensing-Mediated Interferon Response in the Cellular Defense Against Foreign DNA Intrusion.

The introduction of foreign DNA into mammalian cells to express a given gene or genes of interest is a pivotal process with significant implications for molecular biology and gene therapy. Despite the development of various methods to improve transfection efficiency, it remains suboptimal in many cell types. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) senses transfected DNA and elicits an innate immune response, leading to low transfection efficiency. However, the mechanism by which transgene expression is repressed by cGAS-STING activation remains elusive. In this study, we demonstrated the crucial involvement of multiple RNA processing pathways under the control of cGAS-STING-activated IRF3/7 in suppressing transgene expression. These pathways included RNA-sensing genes (e.g., MDA5 and RIGI), as well as the OAS family (mRNA degradation) and the IFIT family (translation inhibition). By depleting IRF3/7, cGAS-STING, or RNA-sensing genes, we observed a significant increase in the transfection efficiency of the treated cells, with the most pronounced effects observed in the STING and MDA5 double-knockdown group. Our findings provide insights into the interconnected roles of DNA- and RNA-sensing mechanisms in innate immune activation triggered by transgene expression, thereby suggesting potential strategies to increase transfection efficiency in biomedical research.