Enhancement of Transgene Expression by the b-Catenin Inhibitor iCRT14

The innate immune system allows cells to detect intracellular pathogen-associated molecular patterns (PAMPs) like endotoxin or cytosolic DNA and then induce inflammation, transcriptional or translational inhibition, or apoptosis in the infected cell. This response is an essential host defense mechanism against viruses and bacteria, but it can also significantly inhibit gene therapy treatments because therapeutic plasmid DNA also triggers an innate immune response. 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). While each of these inhibitors significantly increased transgene (luciferase) expression by at least 2-fold, the b-catenin/TCF4 inhibitor iCRT14 showed the highest enhancement (16 to 35-fold) in multiple cell lines (PC-3, MCF7, & MB49) at a concentration of 1 mM without significantly decreasing cellular proliferation. Alternatively, transgene expression was also enhanced 8-fold by inserting a b-catenin/TCF4 binding motif (TCAAAG) downstream of the EF1a promoter. To further investigate the role of iCRT14 and b-catenin/TCF4 in transgene expression, mRNA-sequencing experiments were conducted to identify host cell genes that were upregulated following transfection and downregulated after the addition of iCRT14. As expected, transfection with plasmid DNA activated the innate immune response and upregulated hundreds (687) of host cell genes, but inhibition of b-catenin/TCF4 with iCRT14 appears to enhance transgene expression by significantly downregulating 22 of those genes (e.g., PTGS2, PLA1A, et al.). Altogether, these results show that inhibition of the innate immune response with SMIs like iCRT14 may be an effective way to improve gene therapy treatments. Overall design: All samples were obtained from PC-3 prostate cancer cells. 3 samples were unmanipulated (controls, C), 3 samples were treated with 1 uM iCRT14 (I), 3 samples were transfected with a polyplex (PEI + pEF-GFP plasmid, P), and 3 samples were transfected and treated with 1 uM iCRT14 (PI) (24 samples total)