Mechanisms of Action of Inhibitory CpG Oilgonucleotides

抑制性 CpG 寡核苷酸的作用机制

• 批准号: 6699378
• 负责人: Robert F Ashman
• 金额: $ 25.81万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6699378
• 关键词: B lymphocyte; apoptosis; binding proteins; cell cycle; free radical oxygen; gel mobility shift assay; immunomodulators; interleukin 6; laboratory mouse; leukocyte activation /transformation; oligonucleotides; synthetic nucleotide; toll like receptor; transcription factor

DESCRIPTION (provided by applicant): Oligonucleotides (ODN) containing the "CpG motif" account for the immune stimulatory activities of bacterial DNA. They are unusually effective mitogens for B cells because, in addition to driving the cells into and through cycle, they also inhibit apoptosis. Certain single base changes in ODN sequence can dramatically reduce activity. We have recently discovered that 3 of the ODN sequence variants actually inhibit cell cycle progress apoptosis protection and IL-6 secretion driven by stimulatory (ST-) ODN, whereas others are weak agonists or neutral in primary B cells. We have also shown that the order of potency of a series of stimulatory (ST-) ODN is the same for cycle entry, apoptosis protection, and IL-6 secretion implying a single control point for ODN signaling. Inhibitory (IN-) ODN block all the biologic effects as well as gene expression and transcription factor activation events (NFkB, AP-1, NF-IL-6) induced by ST-ODN, but not similar events induced by LPS or anti-CD40. Our application will determine the optimal base sequence for inhibition, and estimate the length of ODN sequence recognized by the CpG-recognizing molecule (RM). Spurred by the finding that an ODN with 2 motifs is more potent than ODN with 1, we will test whether bivalence or the sequence of "transplanted" motifs determine the activity of an ODN. Thus we may discover more potent ST- and IN-ODN than those now available. Based on evidence that IN-ODN act proximal to NFkB and AP-1, we will examine earlier events leading to NFkB or AP-1 to locate sites of action of IN-ODN. We have shown that 32P-labeled CpG-ODN bind a series of 4-5 proteins in B cell cytoplasmic extracts, and that the order of avidity of these proteins for ST-ODN matches their order of potency in biologic assays. We will test whether this is also true for IN-ODN, including whether the increased potency associated with having 2 motifs is reflected in avidity for CpG-binding proteins (BP). We will then obtain microsequence data on the most prominent proteins in an attempt to identify them. Toll-Like Receptor (TLR) 9 has recently been shown to be necessary for responses to ODN. Using a TLR9-transfected cell line we will test whether TLR9 or one of its associated proteins binds CpG-ODN directly and if it does, whether the avidity and EMSA mobility match one of the CpG-BPs. The importance of this project lies in the need to develop antidotes for excessive CpG stimulation potentially to be encountered in CpG vaccine trials, and to recognize and avoid IN-ODN motifs when constructing viral vectors for gene therapy and DNA vaccination. Possibly that IN-ODN motifs in mammalian DNA may keep autoimmune responses to endogenous ST-ODN motifs in check.

描述（由申请人提供）：含有“CpG”的寡核苷酸（ODN） 基序”解释了细菌 DNA 的免疫刺激活性。它们是 对 B 细胞异常有效的有丝分裂原，因为除了驱动 细胞进入并完成循环，它们还抑制细胞凋亡。某些单碱基 ODN 序列的变化会显着降低活性。我们最近有 发现 3 个 ODN 序列变体实际上抑制细胞周期 刺激 (ST-) 驱动细胞凋亡保护和 IL-6 分泌 ODN，而其他的在原代 B 细胞中是弱激动剂或中性。我们有 还表明一系列刺激性 (ST-) ODN 的效力顺序为 对于周期进入、细胞凋亡保护和 IL-6 分泌来说也是如此，这意味着 ODN 信令的单一控制点。抑制性 (IN-) ODN 阻断所有 生物效应以及基因表达和转录因子激活 ST-ODN 诱导的事件（NFkB、AP-1、NF-IL-6），但不诱导类似的事件 通过LPS或抗CD40。 我们的应用程序将确定抑制的最佳碱基序列，并且 估计 CpG 识别分子识别的 ODN 序列的长度 （RM）。受到具有 2 个基序的 ODN 比 ODN 更有效这一发现的启发 为 1，我们将测试是否是二价或“移植”基序的序列 确定 ODN 的活性。因此我们可能会发现更有效的 ST- 和 IN-ODN 比现在可用的那些。基于 IN-ODN 作用接近的证据 NFkB 和 AP-1，我们将检查导致 NFkB 或 AP-1 的早期事件来定位 IN-ODN 的作用位点。 我们已经证明 32P 标记的 CpG-ODN 可以结合 B 细胞中的一系列 4-5 个蛋白质 细胞质提取物，并且这些蛋白质的亲合力顺序 ST-ODN 与其在生物测定中的效力顺序相匹配。我们将测试是否 IN-ODN 也是如此，包括效力是否增加 与具有 2 个基序相关的反映在 CpG 结合的亲和力上 蛋白质（BP）。然后我们将获得最突出的微序列数据 蛋白质，试图识别它们。 Toll 样受体 (TLR) 9 有 最近被证明对于响应 ODN 是必要的。使用 TLR9 转染细胞系，我们将测试 TLR9 或其相关之一是否 蛋白质直接结合 CpG-ODN，如果结合，亲合力和 EMSA 是否 移动性与 CpG-BP 之一匹配。该项目的重要性在于 需要开发针对过度 CpG 刺激的解毒剂 CpG 疫苗试验中遇到的情况，并识别和避免 IN-ODN 基序 当构建用于基因治疗和 DNA 疫苗接种的病毒载体时。可能 哺乳动物 DNA 中的 IN-ODN 基序可能保持对内源性的自身免疫反应 ST-ODN 基序受到检查。