Enhancement of Transgene Expression by the b-Catenin Inhibitor iCRT14. 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)

固有免疫系统（innate immune system）可介导细胞识别胞内病原体相关分子模式（pathogen-associated molecular patterns，PAMPs），如内毒素（endotoxin）或胞质DNA（cytosolic DNA），进而在受感染细胞中诱导炎症反应、转录或翻译抑制，乃至细胞凋亡（apoptosis）。该应答是宿主对抗病毒与细菌的核心防御机制，但同时也会显著抑制基因治疗的效果，因为治疗性质粒DNA同样会触发固有免疫应答。
本研究旨在通过小分子抑制剂（small molecule inhibitors，SMIs：iCRT14、姜黄素（curcumin）、氨来呫诺（Amlexanox）、H-151、SC-514及VX-702）抑制固有免疫应答的关键组分，以提升转基因表达水平。尽管上述所有抑制剂均可使转基因（荧光素酶（luciferase））的表达量至少提升2倍，但β-连环蛋白/TCF4抑制剂iCRT14在多种细胞系（PC-3、MCF7及MB49）中、1 mM浓度下展现出最优的增强效果（16至35倍），且未显著降低细胞增殖活性。
此外，在EF1α启动子（EF1α promoter）下游插入β-连环蛋白/TCF4结合基序（TCAAAG），同样可使转基因表达提升8倍。为进一步探究iCRT14及β-连环蛋白/TCF4在转基因表达中的作用机制，本研究开展了mRNA测序（mRNA-sequencing）实验，以筛选转染后上调、经iCRT14处理后下调的宿主细胞基因。正如预期，质粒DNA转染可激活固有免疫应答并上调687个宿主细胞基因；而通过iCRT14抑制β-连环蛋白/TCF4，似乎可通过显著下调其中22个基因（如PTGS2、PLA1A等）来提升转基因表达水平。
综上，本研究结果表明，使用iCRT14这类小分子抑制剂抑制固有免疫应答，或可成为改善基因治疗效果的有效策略。
实验整体设计：所有样本均取自PC-3前列腺癌细胞系。其中3份为未处理的对照组（C组），3份经1 μM iCRT14处理（I组），3份经多聚复合物（聚乙烯亚胺（polyethyleneimine，PEI）+ pEF-GFP质粒）转染（P组），另有3份经转染并以1 μM iCRT14处理（PI组），总计24份样本。