Small Molecule Inhibition of the Innate Immune Response Increases Transgene Expression

Abstract

AbstractForeign molecules like plasmid DNA trigger a complex and potent innate immune response comprised of highly redundant signal transduction cascades that result in the activation of transcription factors and the production of inflammatory cytokines. Unfortunately, this defense mechanism can 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 in prostate (PC3), breast (MCF-7), and murine bladder (MB49) cancer cell lines. 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. 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. These results demonstrate that these inhibitors can improve gene therapy by preventing an inflammatory innate immune response.