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

用于测量染色质相互作用蛋白体内活性的快速而稳健的测定

• 批准号: 10759170
• 负责人: Zu-Wen Sun
• 金额: $ 100.89万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-19 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10759170
• 关键词: Autoimmune; Benchmarking; Binding; Binding Proteins; Biological Assay; Biological Process; Biomedical Research; Cell Line; Cell physiology; Cells; Cellular biology; ChIP-seq; Chromatin; Complex; DNA; Data Analyses; Detection; Development; Disease; Dose; EMSA; Enzyme-Linked Immunosorbent Assay; Equipment; Gene Expression; Gene Expression Regulation; Genomics; Health; Immune; In Situ; Inflammation; Inflammatory; Laboratories; Manuals; Maps; Marketing; Measurement; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacotherapy; Phase; Post-Translational Protein Processing; Proteins; Protocols documentation; Reader; Regulator Genes; Reproducibility; Research; Research Personnel; Response to stimulus physiology; Role; Services; Signal Transduction; Stains; Stimulus; Time; Titrations; Transcript; Validation; Work; assay development; cell type; commercialization; complex data; cost; drug development; drug mechanism; high-throughput drug screening; improved; in vivo; innovation; member; miniaturize; novel; novel therapeutics; programs; research and development; response; transcription factor; transcriptome sequencing

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.

项目摘要 染色质促蛋白（CAP）的活性是基因表达调节的基础。 阐明帽的活性对于理解驱动细胞生物学的调节机制至关重要 健康和疾病的背景，并指导新型治疗剂的发展。但是，这项工作不是 直接，由于CAP活动受到多种因素的调节，包括翻译后修改和 亚细胞定位。因此，研究CAP活动的测定必须特异性检测其活性形式的帽子 （即直接或直接与染色质结合）。基因组映射测定可以提供有关的详细信息 上限参与和本地化；但是，这些测定对于大多数研究人员以及 由于与成本，复杂性相关的高入口屏障，不适合大规模的高通量研究 设备和数据分析。研究上限的其他现有测定法捕获了总细胞丰度（例如， EMSA，ELISA，IHC）或转录级别（例如QPCR，RNA-Seq），实际上不具体量化盖子 与体内染色质有关。因此，需要一个高通量，低成本测定的领域 所有研究人员都可以访问，并且可以通过量化染色质体内参与度来告知CAP活动。 这种测定的市场可用性将通过提供方便来转变为生物医学研究 访问CAP在基因调节程序中的作用和响应，包括药物机制的分析 作用和免疫刺激反应以及高通量药物筛查。 在这里，Epicypher将开发Quanticaptm，这是一个突破性的测定，可以研究体内 盖的活动。该提案的创新是开发有针对性的方法来直接量化 细胞中的盖染色质的总数。我们的方法利用原位免疫目标策略 通过荧光DNA染色结合消费质盖结合的染色质与高度敏感的检测。全面的， 该平台将提供第一个定量，低成本和可扩展的方法来利用帽子的分析 生物医学研究。在直接进入第二阶段研究计划中，我们正在强调平台的发展 研究转录因子（TFS）和染色质读取器蛋白，尽管我们的平台将广泛适用 用于研究任何上限。在第一阶段，我们开发了一个自动化的Quanticap工作流程，证明了可行性 为了使测定在药物治疗后量化CAP的变化，并确定了测定条件以进一步 提高灵敏度。在第二阶段，我们将扩展Quanticap分析开发以覆盖五（5）个高价值目标 使用手动和自动化工作流，包括基因组学验证。我们将验证我们的测定 多种原发性免疫细胞和刺激，然后确定细胞输入阈值，这是一种强大的归一化策略， 和定量测定参数。最后，我们将通过开发来为营销和商业化做准备 Quanticap beta套件并应用自动测定法以进行高影响力的药物开发研究。