Rapid quantification of nuclear citrullination in human neutrophils

快速定量人中性粒细胞核瓜氨酸化

• 批准号: 9911359
• 负责人: Andrea Lynn Johnstone
• 金额: $ 99.35万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-13 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911359
• 关键词: Antibodies; Apoptosis; Arginine; Autoimmune Diseases; Biological Assay; Biological Markers; Blood; Calibration; Cancer Patient; Cell Death; Cells; Charge; Chemicals; Chromatin; Chromatin Structure; Citrulline; Clinical; Clinical Trials; Collection; Cross-Sectional Studies; Development; Disease; Environment; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Exhibits; Feedback; Gene Expression; Generations; Goals; Histones; Human; Lead; Lupus; Malignant Neoplasms; Necrosis; Nuclear; Nucleosomes; Pathology; Patients; Phase; Physiological; Plasma; Post-Translational Protein Processing; Procedures; Recombinants; Recovery; Research; Rheumatoid Arthritis; Sampling; Sepsis; Serotyping; Serum; Severity of illness; Signal Transduction; Source; Symptoms; Testing; Thrombosis; Validation; assay development; biomarker development; biomarker discovery; cancer risk; commercialization; extracellular; innovation; liquid biopsy; neutrophil; next generation; novel; pre-clinical; programs; research and development; sample collection; seropositive

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.

项目摘要 染色质结构和基因表达受组蛋白的翻译后修饰控制 （PTM）在核小体上，染色质的基本重复单元。染色质脱凝仪是部分控制的 通过在组蛋白上将精氨酸转换为瓜氨酸的PAD4依赖性转化（即瓜氨酸化）。值得注意的是，PAD4 中性粒细胞中的激活启动了与细胞凋亡或坏死不同的细胞死亡程序，其中染色质是 在中性粒细胞外陷阱（即netosis）中的细胞中的过度乳腺露硫，反应和挤出。 人们认为将瓜氨酸的染色质释放到血液中直接有助于PAD4相关的病理。 那就是瓜氨酸核小体（cit-nucs）是一系列有前途的血液访问生物标志物 自身免疫性疾病，包括类风湿关节炎（RA），癌症血栓形成和败血症。但是，有 没有能够可靠地量化血清或血浆的核柑桔的测定。 旨在量化cit-Nucs的当前测定 开发和校准。这些类型的标准是有问题的，原因有两个。第一的， 瓜氨酸的组蛋白无法对核体的准确定量，尤其是在低浓度下； 其次，免费组蛋白充满电，并且在血浆中很容易聚集，这显着影响其线性 从血浆样品中恢复。相比之下，我们（和其他）发现核小体在高度稳定 血浆表明这些底物可以为测定定量提供出色的标准。 Epicypher® 正在开创携带生理的重组设计师核小体（DNUC）的商业化 Hisstone PTMS用于下一代表观遗传学测定。在这里，我们正在开发Citnuc™，这是第一个ELISA 准确地量化了用于Netosis研究和临床前生物标志物发展的CIT-NUC。我们的创新性 测定使用重组柠檬奶粉设计师核小体（CIT-DNUC）作为抗体的定量标准 配对选择 /验证和可靠的测定数量。重要的是，与组蛋白不同，Cit-Dnucs可以是 忠实地从血浆样品中恢复了，从而发展了高度敏感的ELISA。 在第一阶段，我们成功使用CIT-DNUC来识别高度特异性的抗体并建立可靠的抗体 定量等离子体中CIT-NUC的标准。我们还检查了密钥生物分析参数并进行了验证 Citnuc Elisa检测健康和RA患者样本之间CIT-NUC差异的能力， 证明我们在临床环境中评估的实用性。在第二阶段，我们将完成生物分析测试 并为Citnuc ELISA套件（AIM 1）定义可靠的释放策略（AIM 1），从而掩盖了商业化的道路。在 目标2和3，我们将开发此测定法对生物标志物发现的临床前应用，重点是RA 和癌症血栓形成，两种与高水平的PAD4依赖性核柑橘粉相关的疾病。 这些目标一起将导致第一代Citnuc ELISA分析的商业发布，该测定法 销售Netosis Research和生物标志物开发应用。