(170c) Enhancement of Gene Therapy Via Inhibition of the Innate Immune Response

While the adaptive immune system detects extracellular pathogens, most cells also have an innate immune system that recognizes intracellular pathogens. For example, when viruses inject their DNA into cells, it is quickly detected by cytosolic â??DNA sensorsâ? that trigger a signaling cascade that leads to the expression of inflammatory cytokines, inhibition of translation, or even apoptosis to prevent the spread of the virus. Unfortunately, this defense mechanism can also significantly inhibit gene therapy, since most methods also introduce therapeutic DNA into the cytoplasm. In this work, we describe several different strategies to improve non-viral gene therapy by inhibiting the innate immune response. Specifically, we show that addition of small molecule inhibitors for several different enzymes (e.g. TLR9, TBK-1, β-catenin, and p38 MAPK) involved in this response can significantly improve transgene (luciferase) expression in cancer cells (e.g. prostate cancer, PC3 cell line). Alternatively, we have also enhanced transgene expression by constructing a plasmid that contains several repeats of a specific DNA sequence that is known to inhibit Toll-Like Receptor 9 (TLR9, a DNA sensor that binds DNA inside endosomes). ELISA assays show that the inhibitors also decrease the expression of inflammatory cytokines (e.g. IL-6), thereby confirming that the enhancement of transgene expression is due to inhibition of the innate immune response. Overall, these results clearly show that the innate immune response is a significant obstacle that must be considered in the development of gene therapy techniques and our work provides multiple alternative strategies to address this issue.