Quantification of combinatorial epigenetic modifications using defined nucleosome standards

使用定义的核小体标准对组合表观遗传修饰进行定量

基本信息

批准号：
10481109
负责人：
Andrea Lynn Johnstone
金额：
$ 102.45万
依托单位：
EPICYPHER, INC.
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10481109
关键词：
Antibodies Automation Binding Biological Assay Cancerous Cell physiology Cells ChIP-seq Chromatin Clinical Clinical Research Code Complex Cytolysis DNA Methylation Data Detection Development Disease Dose Drug Targeting Epigenetic Process Genes Genetic Transcription Genomics Histones Human Human Pathology In Situ Individual Language Literature Malignant Neoplasms Measures Methods Modification Molecular Molecular Conformation Nucleosomes Performance Pharmaceutical Preparations Pharmacologic Substance Pharmacotherapy Phase Plasma Plasma Cells Post-Translational Protein Processing Preparation Protocols documentation Reagent Recombinants Recovery Regulation Research Sampling Services Site-Directed Mutagenesis Specificity Tail Testing Time Validation Work assay development base biomarker development biomarker discovery cancer cell chromatin modification combinatorial cost detection limit drug development drug discovery epigenetic drug genome-wide genomic signature histone modification innovation liquid biopsy novel novel marker response stability testing tool

项目摘要

PROJECT SUMMARY Post-translational modification of histone tails (histone PTMs) and DNA methylation (DNAme) on nucleosomes form a sophisticated molecular code that regulates gene transcription. Aberrant regulation of these chromatin modifications is associated with a vast array of human pathologies. While the majority of work in the field has focused on signatures of individual modifications, combinations of histone PTMs and/or DNAme can be more specific and informative than single marks alone. For instance, although healthy cells and cancerous cells both have H3K27me3 and DNAme distributed genome-wide, the co-localization of these two modifications occurs uniquely in cancer cells. However, existing tools to measure global levels of chromatin modifications are low-throughput, display low sensitivity, and are unable to measure combinatorial modifications (e.g. immunoblot). The development of assays that overcome these limitations and are compatible with multiple sample types (including cellular samples or plasma [for detection of circulating nucleosomes, i.e. liquid biopsy]) will make the study of chromatin modifications widely accessible for academic, clinical, and pharmaceutical research. Here, EpiCypher will develop QuantiNucTM assays, a breakthrough epigenetics platform to quantify single and combinatorial chromatin modifications directly on nucleosomes from cells or plasma samples. The innovation of this proposal includes the a) application of designer nucleosomes (dNucs) to systematically identify top-performing detection reagents and to serve as quantitative assay standards, b) development of recombinant EpiSensors for unbiased detection of DNA and DNAme, and c) development of a proprietary targeted sample processing method for high-throughput cell-based assays. Overall, this platform will provide a quantitative, low- cost, and scalable approach to leverage analysis of chromatin modifications (i.e. histone PTMs and/or DNAme) for chromatin research, drug development, and novel biomarker discovery. In Phase I, we developed a QuantiNuc assay targeting combinatorial H3K4me3+H3K27ac, PTMs that are co-enriched at actively expressed genes. We validated the specificity and performance of this QuantiNuc assay by establishing key analytical parameters and applying the assay to quantify levels of H3K4me3+H3K27ac nucleosomes from human plasma samples. In Phase II, we will develop new QuantiNuc assays to measure other high-value single and combinatorial chromatin modifications and further validate these assays for use with human plasma samples (i.e. liquid biopsy). In addition, we will develop a novel targeted sample processing method for cell-based QuantiNuc assays, which will streamline the process of cell lysis and chromatin fragmentation to deliver a high- throughput, low-cost approach for clinical research. Finally, we will prepare for commercial launch of QuantiNuc assays by assembling beta-kits and performing internal and external validation testing of both liquid biopsy and cell-based assays, which will be used to develop reliable assay protocols and product literature. Market availability of these assays will transform biomarker discovery and accelerate epigenetic drug development.

项目概要组蛋白尾部（组蛋白 PTM）和 DNA 甲基化 (DNAme) 的翻译后修饰核小体形成复杂的分子密码来调节基因转录。这些监管失当染色质修饰与多种人类病理学相关。虽然大部分工作都在该领域的重点是个体修饰的特征，组蛋白 PTM 和/或 DNAme 的组合可以比单独的单个标记更具体、信息更丰富。例如，虽然健康细胞和癌细胞细胞均具有分布在全基因组范围内的 H3K27me3 和 DNAme，这两种修饰的共定位独特地发生在癌细胞中。然而，测量染色质修饰全局水平的现有工具是低通量，显示低灵敏度，并且无法测量组合修饰（例如免疫印迹）。克服这些限制并与多种样本类型兼容的检测方法的开发（包括细胞样本或血浆[用于检测循环核小体，即液体活检]）将使染色质修饰的研究广泛应用于学术、临床和药物研究。在这里，EpiCypher 将开发 QuantiNucTM 检测，这是一个突破性的表观遗传学平台，用于量化单个以及直接对细胞或血浆样品的核小体进行组合染色质修饰。这该提案的创新包括a）应用设计核小体（dNucs）来系统地识别顶级性能的检测试剂并作为定量测定标准，b) 重组的开发 EpiSensors 用于公正地检测 DNA 和 DNAme，以及 c) 开发专有的目标样品高通量细胞分析的处理方法。总体而言，该平台将提供定量的、低成本和可扩展的方法来利用染色质修饰分析（即组蛋白 PTM 和/或 DNAme）用于染色质研究、药物开发和新型生物标志物发现。在第一阶段，我们开发了 QuantiNuc 检测针对组合 H3K4me3+H3K27ac，在活跃表达处共富集 PTM 基因。我们通过建立关键分析验证了该 QuantiNuc 检测的特异性和性能参数并应用该测定来定量人血浆中 H3K4me3+H3K27ac 核小体的水平样品。在第二阶段，我们将开发新的 QuantiNuc 检测方法来测量其他高价值的单一和组合染色质修饰并进一步验证这些检测方法是否可用于人类血浆样本（即液体活检）。此外，我们将开发一种新颖的基于细胞的靶向样品处理方法 QuantiNuc 检测，将简化细胞裂解和染色质碎片的过程，以提供高高通量、低成本的临床研究方法。最后，我们将为QuantiNuc 的商业发布做准备通过组装 beta 套件并对液体活检和进行内部和外部验证测试来进行测定基于细胞的测定，将用于开发可靠的测定方案和产品文献。市场这些测定的可用性将改变生物标志物的发现并加速表观遗传药物的开发。