Rapid quantification of nuclear citrullination in human neutrophils

项目摘要 组蛋白翻译后修饰控制染色质结构和基因表达 核小体上的核转录修饰（PTMs）是染色质的基本重复单位。染色质去浓缩部分地受到控制 通过组蛋白（即瓜氨酸）上精氨酸向瓜氨酸的PAD 4依赖性转化。值得注意的是，PAD 4 嗜中性粒细胞中的活化启动不同于凋亡或坏死的细胞死亡程序，其中染色质是 高瓜氨酸化、去致密化，并在中性粒细胞胞外陷阱中从细胞中挤出（即NETosis）。的 瓜氨酸化染色质释放到血液中被认为直接导致PAD 4相关的病理。 因此，瓜氨酸化核小体（Cit-Nucs）是一种有前途的血液可及生物标志物，用于一系列疾病。 自身免疫性疾病，包括类风湿性关节炎（RA）、癌症血栓形成和败血症。但有 没有能够可靠地定量血清或血浆中的核小体瓜氨酸的测定法。 旨在定量Cit-Nucs的当前测定使用瓜氨酸化组蛋白作为测定的标准品。 开发和校准。这类标准有两个主要原因：第一， 瓜氨酸化组蛋白不能提供核小体的准确定量，特别是在低浓度下; 第二，游离组蛋白是高度带电的，并且容易在血浆中聚集，这显著影响它们的线性。 血浆样品的回收率。相比之下，我们（和其他人）已经发现核小体在 血浆，表明这些底物可提供用于测定定量的上级标准。EpiCypher® 是携带生理活性的重组设计核小体（dNucs）商业化的先驱。 用于下一代表观遗传学分析的组蛋白PTM。在这里，我们正在开发CitNuc™，这是第一个ELISA， 准确定量Cit-Nucs用于NETosis研究和临床前生物标志物开发。我们的创新 测定使用重组瓜氨酸化设计者核小体（Cit-dNucs）作为抗体的定量标准 配对选择/验证和可靠的测定定量。重要的是，与组蛋白不同，Cit-dNucs可以是 从血浆样品中忠实地回收，使得能够开发高度灵敏的ELISA。 在第一阶段，我们成功使用Cit-dNucs识别高度特异性抗体并建立可靠的 用于定量血浆中Cit-Nucs的标准品。我们还检查了关键的生物分析参数， CitNuc ELISA检测健康和RA患者样品之间Cit-Nucs差异的能力， 证明了我们的分析在临床环境中的实用性。在第二阶段，我们将完成生物分析测试， 并为CitNuc ELISA试剂盒制定可靠的批放行策略（目标1），为商业化铺平道路。在 目标2和3，我们将开发这种检测方法的临床前应用，用于生物标志物发现，重点是RA 和癌症血栓形成，这两种疾病与高水平的PAD 4依赖性核小体瓜氨酸有关。 总之，这些目标将导致第一代CitNuc ELISA检测试剂盒的商业化推出， 用于NETosis研究和生物标志物开发应用。

PROJECT SUMMARY Chromatin structure and gene expression are controlled by histone post-translational modifications (PTMs) on nucleosomes, the basic repeating unit of chromatin. Chromatin decondensation is controlled in part by the PAD4-dependent conversion of arginine to citrulline on histones (i.e. citrullination). Significantly, PAD4 activation in neutrophils initiates a cell death program distinct from apoptosis or necrosis, wherein chromatin is hypercitrullinated, decondensed, and extruded from the cell in neutrophil extracellular traps (i.e. NETosis). The release of citrullinated chromatin into the blood is thought to contribute directly to PAD4-related pathologies. Thus, citrullinated nucleosomes (Cit-Nucs) are a promising blood-accessible biomarker for a range of autoimmune diseases, including rheumatoid arthritis (RA), cancer thrombosis, and sepsis. However, there are no assays capable of reliably quantifying nucleosome citrullination from serum or plasma. Current assays that aim to quantify Cit-Nucs use citrullinated histones as standards for assay development and calibration. These type of standards are problematic for two major reasons; First, citrullinated histones fail to provide accurate quantification of nucleosomes, especially at low concentrations; second, free histones are highly charged and readily aggregate in plasma, which significantly impacts their linear recovery from plasma samples. By contrast, we (and others) have found that nucleosomes are highly stable in plasma, suggesting that these substrates may provide superior standards for assay quantification. EpiCypher® is pioneering the commercialization of recombinant designer nucleosomes (dNucs) carrying physiological histone PTMs for next-generation epigenetics assays. Here, we are developing CitNuc™, the first ELISA to accurately quantify Cit-Nucs for NETosis research and preclinical biomarker development. Our innovative assay uses recombinant citrullinated designer nucleosomes (Cit-dNucs) as quantification standards for antibody pair selection / validation and reliable assay quantification. Importantly, unlike histones, Cit-dNucs can be faithfully recovered from plasma samples, enabling development of a highly sensitive ELISA. In Phase I, we successfully used Cit-dNucs to identify highly specific antibodies and establish reliable standards for quantification of Cit-Nucs in plasma. We also examined key bioanalytical parameters and validated the ability of CitNuc ELISA to detect differences in Cit-Nucs between healthy and RA patient samples, demonstrating the utility of our assay in a clinical environment. In Phase II, we will complete bioanalytical testing and define reliable lot-release strategies for CitNuc ELISA kits (Aim 1), paving the way for commercialization. In Aims 2 and 3, we will develop the preclinical application of this assay for biomarker discovery, focusing on RA and cancer thrombosis, two diseases associated with high levels of PAD4-dependent nucleosome citrullination. Together, these Aims will result in the commercial launch of a first-generation CitNuc ELISA assay, which will be marketed for NETosis research and biomarker development applications.