Villanova University, Department of Chemical & Biological Engineering, United States.
Villanova University, Department of Chemical & Biological Engineering, United States.
Plasmid. 2021 Mar;114:102556. doi: 10.1016/j.plasmid.2021.102556. Epub 2021 Jan 17.
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.
先天免疫反应是一种重要的防御机制，使细胞能够检测到病原体相关分子模式（PAMPs），如内毒素或细胞质 DNA，然后诱导防御基因的表达，限制病毒和其他病原体的复制。然而，一些基因治疗中使用的治疗性 DNA 也可以触发先天免疫反应，激活宿主细胞基因，可能抑制转基因的表达。本研究的目的是通过使用小分子抑制剂（iCRT14、姜黄素、Amlexanox、H-151、SC-514 和 VX-702）抑制先天免疫反应的关键成分来增强转基因表达。大多数抑制剂至少将转基因（荧光素酶）表达提高了 2 倍，但β-catenin/TCF4 抑制剂 iCRT14 在多种细胞系（PC-3、MCF7 和 MB49）中显示出最高的增强作用（16 至 35 倍），而不会显著降低细胞增殖。或者，将β-catenin/TCF4 结合基序（TCAAAG）克隆到 EF1α 启动子中也可以将转基因表达提高 8 倍。为了进一步研究β-catenin/TCF4 在转基因表达中的作用，进行了 mRNA 测序实验，以鉴定在用 PEI 转染后上调但在用 iCRT14 处理后下调的宿主细胞基因。正如预期的那样，用质粒 DNA 转染激活了先天免疫反应，并上调了数百个（687 个）防御基因，但只有 7 个基因在 iCRT14 的存在下下调（例如，PTGS2 和 PLA1A）。总之，这些结果表明，通过使用小分子抑制剂（如 iCRT14）抑制先天免疫反应，可以增强转基因表达，iCRT14 抑制β-catenin/TCF4 以防止特定宿主细胞基因的表达。
