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

• 批准号: 10630256
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630256
• 关键词: Acceleration; Antibodies; Automation; Binding; Biological Assay; Cancerous; Cell physiology; Cells; ChIP-seq; Chromatin; Clinical; Clinical Research; Code; Complex; Cytolysis; DNA; 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; Marketing; Measures; Methods; Modification; Molecular; Molecular Conformation; Nucleosomes; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacotherapy; Phase; Plasma; Post-Translational Protein Processing; Preparation; Protocols documentation; Reagent; Recombinants; Recovery; Regulation; Research; Sampling; Services; Site-Directed Mutagenesis; Specificity; Tail; Testing; Time; Validation; Western Blotting; Work; assay development; biomarker development; biomarker discovery; cancer cell; chromatin modification; combinatorial; commercial launch; 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

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.

项目摘要 组蛋白尾巴（组蛋白PTM）和DNA甲基化（DNAME）的翻译后修饰 核小体形成了调节基因转录的复杂分子代码。对这些的异常调节 染色质修饰与各种各样的人类病理有关。而大多数工作 字段专注于单个修改，组蛋白PTM和/或dname的组合的签名 与单个标记相比，要更具体和信息丰富。例如，尽管健康细胞和癌细胞 细胞都具有H3K27ME3和DNAME分布的全基因组，这两种修饰的共定位 在癌细胞中独特地发生。但是，现有的测量染色质修饰全球水平的工具是 低通量，表现出低灵敏度，并且无法测量组合修饰（例如免疫印迹）。 克服这些局限性并与多种样本类型兼容的测定的开发 （包括细胞样品或血浆[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可用于学术，临床和药物研究。 在这里，Epicypher将开发量化分析，这是一个量化单个的突破性表观遗传学平台 直接对细胞或血浆样品的核小体进行了组合染色质修饰。这 该提案的创新包括a）设计器核小体（DNUC）的应用 表现最佳检测试剂并用作定量测定标准，b）重组的发展 无偏见的DNA和DNAME的情节，以及c）开发专有的靶向样品 高通量基于细胞的测定方法的处理方法。总体而言，该平台将提供一个定量，低 - 成本和可扩展的方法来利用染色质修饰的分析（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分析靶向组合H3K4ME3+H3K27AC，PTM，在积极表达的 基因。我们通过建立关键分析来验证了该量子分析的特异性和性能 参数并应用于量化人血浆中H3K4me3+H3K27AC核小体的水平 样品。在II阶段，我们将开发新的Quantinuc分析，以测量其他高价值单一的单一分析和 组合染色质修饰并进一步验证这些测定法以与人血浆样品一起使用 （即液体活检）。此外，我们将开发一种针对基于细胞的新型靶向样品处理方法 Quantinuc测定法，该测定将简化细胞裂解和染色质碎片的过程，以提供高 用于临床研究的吞吐量，低成本方法。最后，我们将为Quantinuc的商业推出做准备 通过组装β套和进行液体活检和外部验证测试的测定 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发现并加速表观遗传药物的开发。