Rapid and robust assay for measurement of in vivo activity of chromatin-interacting proteins

项目摘要 染色质相关蛋白（CAP）的活性是基因表达调控的基础。 阐明CAP活性对于理解驱动细胞生物学的调节机制至关重要，无论是在细胞内还是在细胞外， 健康和疾病的背景，并指导新疗法的开发。然而，这项工作并不是 直接，因为CAP活性受多种因素调节，包括翻译后修饰和 亚细胞定位。因此，研究CAP活性的测定必须特异性地检测活性形式的CAP (i.e.,直接或复合结合于染色质）。基因组作图测定可以提供关于以下方面的详细信息： CAP参与和定位;然而，这些测定法对于大多数研究人员来说是不可访问的， 由于与成本，复杂性， 设备和数据分析。研究CAP的其他现有测定捕获总细胞丰度（例如， EMSA、ELISA、IHC）或转录水平（例如，qPCR、RNA-seq），并且实际上不特异性地定量CAP 与体内染色质相关。因此，现场需要一种高通量、低成本的测定法， 所有研究人员都可以使用，并且可以通过量化染色质上的体内参与来告知CAP活性。 这样的测定的市场可用性将通过提供方便而对生物医学研究具有变革性。 获得研究CAP在基因调控计划中的作用和反应，包括分析药物机制 作用和免疫刺激反应，以及高通量药物筛选。 在这里，EpiCypher将开发QuantiCAPTM，这是一种突破性的检测方法，可以在体内研究 CAP的活动。该提案的创新之处在于制定了一种有针对性的方法， 细胞中CAP结合染色质的总量。我们的方法利用原位免疫靶向策略 切除CAP结合的染色质，并结合荧光DNA染色的高灵敏度检测。总的来说， 该平台将提供第一个定量、低成本和可扩展的方法，以利用CAP分析， 生物医学研究在这个直接进入第二阶段的研究计划中，我们强调了平台的开发 研究转录因子（TF）和染色质阅读器蛋白，尽管我们的平台将广泛适用 任何CAP的研究。在第一阶段，我们开发了一个自动化的QuantiCAP工作流程，证明了其可行性。 对于定量药物治疗后CAP变化的测定，并确定了进一步 提高灵敏度。在第二阶段，我们将扩展QuantiCAP检测开发，以涵盖五（5）个高价值靶标 使用手动和自动化工作流程，包括基因组学验证。我们将验证我们的检测方法， 不同的原代免疫细胞和刺激，然后确定细胞输入阈值，一个强大的标准化策略， 和定量测定参数。最后，我们将通过开发 QuantiCAP beta试剂盒，并应用自动化测定进行高影响力的药物开发研究。

PROJECT SUMMARY The activity of chromatin-associating proteins (CAPs) is fundamental to regulation of gene expression. Elucidating CAP activity is central to understanding the regulatory mechanisms that drive cell biology, both in the context of health and disease, and to guide development of novel therapeutics. However, this work is not straightforward, as CAP activity is regulated by multiple factors including post-translational modifications and subcellular localization. Thus, assays to study CAP activity must specifically detect CAPs in their active form (i.e., bound to chromatin, directly or in complex). Genomic mapping assays can provide detailed information on CAP engagement and localization; however, these assays are inaccessible to a majority of researchers and unsuitable for large-scale, high-throughput studies due to a high barrier of entry associated with cost, complexity, equipment, and data analysis. Other existing assays to study CAPs capture total cellular abundance (e.g., EMSAs, ELISAs, IHC) or transcript levels (e.g., qPCR, RNA-seq), and do not specifically quantify CAPs actually associated with chromatin in vivo. Thus, there is a field need for a high-throughput, low-cost assay that is accessible to all researchers, and can inform CAP activity via quantification of in vivo engagement on chromatin. Market availability of such an assay would be transformative for biomedical research by providing convenient access to study the role and response of CAP in gene regulatory programs, including analysis of drug mechanism of action and immune stimulus responses, as well as for high-throughput drug screening. Here, EpiCypher will develop QuantiCAPTM, a breakthrough assay that will enable the study of in vivo activity of CAPs. The innovation of this proposal is the development of a targeted approach to directly quantify the total amount of CAP-bound chromatin in cells. Our approach leverages an in situ immunotargeting strategy to excise CAP-bound chromatin combined with highly sensitive detection by a fluorescent DNA stain. Overall, this platform will provide the first quantitative, low-cost, and scalable approach to leverage analysis of CAPs for biomedical research. In this Direct to Phase II research program, we are highlighting development of the platform to study transcription factors (TFs) and chromatin reader proteins, though our platform will be broadly applicable for the study of any CAP. In Phase I, we developed an automated QuantiCAP workflow, demonstrating feasibility for the assay to quantify changes in a CAP following drug treatment, and identified assay conditions to further improve sensitivity. In Phase II, we will extend QuantiCAP assay development to cover five (5) high value targets using both manual and automated workflows, including genomics validation. We will validate our assays across diverse primary immune cells and stimuli, then determine cell input thresholds, a robust normalization strategy, and quantitative assay parameters. Finally, we will prepare for marketing and commercialization by developing a QuantiCAP beta kit and applying the automated assay to perform high-impact drug development studies.