Small Molecule Inhibition of the Innate Immune Response Increases Transgene Expression

Upon recognition of foreign molecules such as plasmid DNA, the innate immune system triggers a complex and potent response comprised of highly redundant signal transduction cascades that result in the activation of transcription factors and the production of inflammatory cytokines. The cytokines then stimulate specific genes that protect the cell from pathogens by inducing apoptosis or inhibiting translation. Unfortunately, this defense mechanism can also hinder gene therapy by inhibiting transgene expression. The goal of this study was to increase transgene expression by inhibiting key components of the innate immune response (β-catenin, NF-κB/AP1, TBK1, TLR9, and p38 MAPK) with small molecule inhibitors (iCRT-14, curcumin, BX-795, E6446, and VX-702 respectively). The effects of each drug on transgene (luciferase) expression, inflammatory cytokine (IL-6) levels, and cell viability were quantified. Each of these inhibitors was able to increase transgene expression in prostate (PC3), breast (MCF-7), and murine bladder (MB49) cancer cell lines. 

Overall, the β-catenin inhibitor iCRT-14 (1 μM) provided the highest enhancement of 35.5 ± 19-fold in MCF-7 cells, while the other inhibitors increased transgene expression at a more modest level (2-9 fold). The optimal concentrations of iCRT-14, curcumin, and VX-702 showed no significant effect on cell proliferation; however, optimal concentrations of BX-795 and E6446 did significantly reduce cell proliferation. Combinations of the SMIs were also tested, but no additive effect on transgene expression was observed. Nonetheless, inhibition of the innate immune response by iCRT-14 and curcumin was confirmed by a concomitant decrease in IL-6 production in PC3 cells. Overall, these results demonstrate that these inhibitors can improve gene therapy by preventing an inflammatory innate immune response.