Mechanisms of Action of Inhibitory CpG Oligonucleotides

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。他们是 对B细胞来说是非常有效的有丝分裂原，因为除了驱动 细胞进入和通过周期，他们也抑制凋亡。某些单碱基 ODN序列的改变可显著降低活性。我们最近 发现3种ODN序列变体实际上抑制细胞周期 由刺激性（ST-）驱动的细胞凋亡保护和IL-6分泌 ODN，而其他在原代B细胞中是弱激动剂或中性的。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、凋亡保护和IL-6分泌也是如此，这意味着 用于ODN信令的单个控制点。抑制性（IN-）ODN阻断所有的 生物学效应以及基因表达和转录因子激活 ST-ODN诱导的NFkB、AP-1、NF-IL-6事件，但不诱导类似事件 LPS或抗CD40抗体。 我们的应用程序将确定抑制的最佳碱基序列， 估算CpG识别分子识别的ODN序列长度 （RM）。由于发现具有2个基序的ODN比ODN更有效， 对于1，我们将测试是否二价或"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 比现在的那些更好。基于IN-ODN作用于近端的证据， NFkB和AP-1，我们将检查导致NFkB或AP-1的早期事件以定位 IN-ODN的作用位点。 我们已经证明，32 P标记的CpG-ODN结合B细胞中的一系列4 - 5种蛋白 细胞质提取物，这些蛋白质的亲合力的顺序， ST-ODN与其在生物测定中的效力顺序相匹配。我们将测试 对于IN-ODN也是如此，包括增加的效力是否 与具有2个基序相关的是反映在对CpG结合的亲合力中 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具有 最近被证明是必要的响应ODN。使用 TLR 9转染的细胞系我们将测试TLR 9或其相关之一是否 蛋白质直接结合CpG-ODN，如果结合，则是否亲和力和EMSA 移动性与其中一个CpG-BP匹配。这个项目的重要性在于 需要开发针对过度CpG刺激的解毒剂，这可能是 在CpG疫苗试验中遇到，并识别和避免IN-ODN基序 用于构建基因治疗和DNA疫苗接种的病毒载体。可能 哺乳动物DNA中的IN-ODN基序可以保持对内源性免疫应答， ST-ODN基序已检查。

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.