Rapid quantification of nuclear citrullination in human neutrophils

快速定量人中性粒细胞核瓜氨酸化

基本信息

批准号：
10331838
负责人：
Andrea Lynn Johnstone
金额：
$ 97.47万
依托单位：
EPICYPHER, INC.
依托单位国家：
美国
项目类别：
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/10331838
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

项目摘要

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.

项目概要染色质结构和基因表达受组蛋白翻译后修饰控制核小体上的翻译后修饰 (PTM)，染色质解缩的基本重复单位受到部分控制。通过组蛋白上的 PAD4 依赖性精氨酸转化为瓜氨酸（即瓜氨酸化）。中性粒细胞的激活会启动与细胞凋亡或坏死不同的细胞死亡程序，而染色质则过度瓜氨酸化、解凝并从中性粒细胞胞外陷阱中的细胞中挤出（即 NETosis）。瓜氨酸染色质释放到血液中被认为直接导致 PAD4 相关病理。因此，瓜氨酸核小体 (Cit-Nucs) 是一种有前景的血液可及性生物标志物，可用于多种疾病的检测。然而，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。没有能够可靠地定量血清或血浆中核小体瓜氨酸化的测定方法。当前旨在量化 Cit-Nucs 的检测使用瓜氨酸组蛋白作为检测标准这些类型的标准存在问题主要有两个原因：瓜氨酸组蛋白无法准确定量核小体，尤其是在低浓度下；其次，游离组蛋白带高电荷，很容易在血浆中聚集，这显着影响其线性相比之下，我们（和其他人）发现核小体在血浆样品中高度稳定。血浆，表明这些底物可以为 EpiCypher® 测定提供优异的标准。是携带生理学的重组设计核小体 (dNucs) 商业化的先驱用于下一代表观遗传学检测的组蛋白 PTM 在这里，我们正在开发 CitNuc™，这是第一个用于下一代表观遗传学检测的 ELISA。准确量化 Cit-Nucs，用于 NETosis 研究和临床前生物标志物开发。测定使用重组瓜氨酸设计核小体 (Cit-dNucs) 作为抗体的定量标准配对选择/验证和测定可靠的定量重要的是，与组蛋白不同，Cit-dNucs 可以是忠实地从血浆样品中回收，从而能够开发高灵敏度的 ELISA。在第一阶段，我们成功地使用 Cit-dNucs 来识别高度特异性的抗体并建立可靠的我们还检查了关键的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-Nucs 差异的能力，在第二阶段，我们将完成生物分析测试，展示我们的检测方法在临床环境中的实用性。并为 CitNuc ELISA 试剂盒制定可靠的批签发策略（目标 1），为商业化铺平道路。目标 2 和 3，我们将开发该检测方法的临床前应用，以发现生物标志物，重点关注 RA 和癌症血栓形成，这两种疾病与高水平的 PAD4 依赖性核小体瓜氨酸化有关。这些目标共同推动第一代 CitNuc ELISA 检测的商业化推出，这将上市用于 NETosis 研究和生物标志物开发应用。