The innate immune response is an essential defense mechanism that allows cells to detect pathogen-associated molecular patterns (PAMPs) like endotoxin or cytosolic DNA and then induce the expression of defensive genes that restrict the replication of viruses and other pathogens. However, the therapeutic DNA used in some gene therapy treatments can also trigger the innate immune response, which activates host cell genes that may inhibit transgene expression. The goal of this study was to enhance transgene expression by inhibiting key components of the innate immune response with small molecule inhibitors (iCRT14, curcumin, Amlexanox, H-151, SC-514, & VX-702). Most of the inhibitors significantly increased transgene (luciferase) expression at least 2-fold, but the β-catenin/TCF4 inhibitor iCRT14 showed the highest enhancement (16 to 35-fold) in multiple cell lines (PC-3, MCF7, & MB49) without significantly decreasing cellular proliferation. Alternatively, cloning a β-catenin/TCF4 binding motif (TCAAAG) into the EF1α promoter also enhanced transgene expression up to 8-fold. To further investigate the role of β-catenin/TCF4 in transgene expression, mRNA-sequencing experiments were conducted to identify host cell genes that were upregulated following transfection with PEI but down-regulated after the addition of iCRT14. As expected, transfection with plasmid DNA activated the innate immune response and upregulated hundreds (687) of defensive genes, but only 7 of those genes were down-regulated in the presence of iCRT14 (e.g., PTGS2 & PLA1A). Altogether, these results show that transgene expression can be enhanced by inhibiting the innate immune response with SMIs like iCRT14, which inhibits β-catenin/TCF4 to prevent the expression of specific host cell genes.
Keywords: Innate immune response; Non-viral gene delivery; PEI; TCF4; iCRT14; β-Catenin.
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