Mechanism of action and therapeutic utility of immunosuppressive oligonucleotide

免疫抑制寡核苷酸的作用机制和治疗用途

• 批准号: 8552865
• 负责人: Dennis Klinman
• 金额: $ 37.49万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552865
• 关键词: ATF2 gene; Animals; Anti-Inflammatory Agents; Antigens; Autoimmunity; Binding; Binding Sites; Bladder; CEBPA gene; CREB1 gene; Carcinogens; Cells; Chronic; DNA; DNA Sequence; DNA-Binding Proteins; Development; Disease; Epidemiologic Studies; Evaluation; Frequencies; Gastrointestinal tract structure; Gene Expression; Genes; Genetic Transcription; Immune; Immune response; Immunosuppressive Agents; In Vitro; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Injury; Leukocytes; Life; Ligands; Link; Liver; Lung diseases; MAP Kinase Gene; MAPK1 gene; MAPK14 gene; MAPK3 gene; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Messenger RNA; Microarray Analysis; Modeling; Morbidity - disease rate; Mus; Neoplasm Metastasis; Nucleic Acid Regulatory Sequences; Oligonucleotides; Pathway interactions; Pattern; Pneumonia; Predisposition; Prevention; Production; Property; Regulation; Regulator Genes; Reproductive system; Research; Research Design; Risk; Septic Shock; Signal Pathway; Silicon Dioxide; Silicosis; Skin Carcinogenesis; Study models; Systems Biology; Therapeutic; Tissues; angiogenesis; bZIP Domain; bZIP Protein; cancer cell; cell type; cellular targeting; chemokine; cigarette smoking; cytokine; dimethylbenzanthracene; in vivo; information gathering; interest; mortality; novel; receptor; response; stem; transcription factor; tumor; tumor progression; tumorigenesis; uptake

Synthetic oligonucleotides (ODN) expressing repetitive TTAGGG motifs patterned after hexameric sequences present at high frequency in mammalian teleomeres down-regulate the inflammatory immune responses elicited by a broad range of TLR ligands and the adaptive immune cell responses induced by polyclonal activators and antigens. These suppressive ODN are useful in the treatment of diseases characterized by over-exuberant immune responses, including septic shock and autoimmunity. Since joining the NCI we demonstrated that suppressive ODN are also useful in the prevention/treatment of the life-threatening inflammation caused by silicosis. Specifically, suppressive ODN significantly reduced mortality and morbidity in a murine silicosis model. We recently developed a novel model in which a combination of silica plus NNK (the major carcinogen present in cigarette smoke) increase the risk of lung cancer in mice. Recent results suggest that systemically administered suppressive ODN alter the host immune milieu. We are harnessing this effect to reduce host susceptibility to inflammation-induced cancer. The major focus of our ongoing research involves examining the effect of Sup ODN in the DMBA/TPA model of skin carcinogenesis and the novel model of silica plus NNK induced lung cancer. We find that Sup ODN significantly reduce both the number of mice that develop tumors and the number of tumors/animal in these murine models of inflammation-associated tumorigenesis. To clarify the mechanism by which Sup ODN inhibit tumor development, various measures of inflammation were examined. In both models studied, leukocyte infiltration and the production of pro-inflammatory cytokines and chemokines were significantly reduced whereas control ODN had no significant effect. We are extending these studies to include other agents that cause pulmonary inflammation and disease. We are also using microarray technology to identify the genes and regulatory networks triggered by suppressive ODN. These microarray studies indicate that very large numbers of genes are rapidly down-regulated following the administration of suppressive ODN. Two mechanisms by which Sup ODN broadly reduce gene expression are the focus of ongoing evaluation: i) that Sup ODN target TTAGGG and/or CCCTAA motifs present in the regulatory regions of critical genes (such as MAPKs) and ii) that Sup ODN inhibit the activity of bZIP proteins (regulatory DNA binding proteins that share a basic leucine zipper domain). In the context of MAPK regulation, mRNA encoding MAPK1, MAPK3 and MAPK14 (which stimulate the ERK-2, ERK-3 and p38 dependent pathways, respectively), as well as the transcription factors they regulate (ATF2, CREB1, NFKB1), contain TTAGGG and/or CCCTAA motifs in their regulatory regions that could be targeted by Sup ODN via an anti-sense mechanism . Since MAPKs exert a stabilizing effect on mRNAs encoding multiple inflammatory genes, down-regulating their expression could have a broad effect on the innate immune response. bZIP proteins are transcription factors that broadly influence gene expression. Transcription binding site analysis shows that many genes down-regulated by Sup ODN contain regulatory domains recognized by bZIP proteins (e.g. CREB1, CEBPA, and FOS). Indeed, 79% of the components in the TLR9 signaling pathway regulated by bZIP proteins are significantly down-regulated by Sup ODN (p < .0001). We examined whether Sup ODN inhibited the binding of bZIP proteins to their target DNA sequences and found that Sup ODN selectively inhibited the binding of three different classes of bZIP protein (CREB, C/EBPa and MAFg) to their target DNAs.Information gathered on the targets and mechanism(s) of action of suppressive ODN will support studies designed to explore their therapeutic utility.

表达重复TTAGGG基序的合成寡核苷酸（ODN）在哺乳动物端粒中以高频率存在的六聚体序列之后图案化，下调由广泛的TLR配体引起的炎性免疫应答和由多克隆活化剂和抗原诱导的适应性免疫细胞应答。 这些抑制性ODN可用于治疗以过度旺盛的免疫应答为特征的疾病，包括败血性休克和自身免疫。 自从加入NCI以来，我们证明了抑制性ODN在预防/治疗由硅肺引起的危及生命的炎症中也是有用的。 具体而言，抑制性ODN显著降低了小鼠硅肺模型的死亡率和发病率。 我们最近开发了一种新的模型，其中二氧化硅和NNK（香烟烟雾中存在的主要致癌物质）的组合增加了小鼠患肺癌的风险。 最近的研究结果表明，全身施用抑制性ODN改变宿主的免疫环境。 我们正在利用这种效应来降低宿主对炎症诱导的癌症的易感性。 我们正在进行的研究的主要焦点包括检查Sup ODN在皮肤癌发生的DMBA/TPA模型和二氧化硅加NNK诱导的肺癌的新模型中的作用。 我们发现，Sup ODN显着减少小鼠的数量，发展肿瘤和肿瘤/动物在这些小鼠模型中的炎症相关的肿瘤发生的数量。为了阐明Sup ODN抑制肿瘤发展的机制，检查了炎症的各种测量。 在研究的两种模型中，白细胞浸润和促炎细胞因子和趋化因子的产生显著减少，而对照ODN没有显著影响。我们正在扩展这些研究，以包括其他引起肺部炎症和疾病的药物。我们也在使用微阵列技术来鉴定由抑制性ODN触发的基因和调控网络。 这些微阵列研究表明，在施用抑制性ODN后，非常大量的基因被迅速下调。 Sup ODN广泛降低基因表达的两种机制是正在进行的评估的焦点：i）Sup ODN靶向关键基因（如MAPK）调控区中存在的TTAGGG和/或CCCTAA基序，ii）Sup ODN抑制bZIP蛋白（共享碱性亮氨酸拉链结构域的调控DNA结合蛋白）的活性。 在MAPK调控的背景下，编码MAPK 1、MAPK 3和MAPK 14（分别刺激ERK-2、ERK-3和p38依赖性途径）的mRNA以及它们调控的转录因子（ATF 2、CREB 1、NFKB 1）在其调控区域中含有TTAGGG和/或CCCTAA基序，其可通过反义机制被Sup ODN靶向。 由于MAPKs对编码多种炎症基因的mRNA发挥稳定作用，因此下调其表达可能对先天免疫反应产生广泛影响。bZIP蛋白是广泛影响基因表达的转录因子。 转录结合位点分析表明，许多被Sup ODN下调的基因含有bZIP蛋白识别的调控结构域（如CREB 1、CEBPA和FOS）。 事实上，79%的TLR 9信号通路中由bZIP蛋白调节的组分被Sup ODN显著下调（p 0.0001）。 我们检测了Sup ODN是否抑制bZIP蛋白与其靶DNA序列的结合，发现Sup ODN选择性地抑制了三种不同类型的bZIP蛋白（CREB、C/EBPa和MAFg）与其靶DNA的结合，为进一步研究Sup ODN的作用靶点和机制提供了理论依据。