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.

项目概要 染色质相关蛋白 (CAP) 的活性对于基因表达的调节至关重要。 阐明 CAP 活性对于理解驱动细胞生物学的调控机制至关重要，无论是在 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 很简单，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 活性的测定必须专门检测活性形式的 CAP （即直接或复合地结合到染色质）。基因组作图分析可以提供详细信息 CAP 参与和本地化；然而，大多数研究人员无法进行这些测定 由于与成本、复杂性相关的高进入壁垒，不适合大规模、高通量研究， 设备、数据分析。其他现有的研究 CAP 的方法可捕获总细胞丰度（例如， EMSA、ELISA、IHC）或转录水平（例如 qPCR、RNA-seq），并且实际上并不具体量化 CAP 与体内染色质相关。因此，现场需要一种高通量、低成本的检测方法 所有研究人员都可以访问，并且可以通过量化体内染色质参与来了解 CAP 活性。 这种检测方法的市场可用性将通过提供便捷的方式为生物医学研究带来变革。 获得研究 CAP 在基因调控项目中的作用和反应的机会，包括药物机制分析 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，可以实现体内研究 CAP 的活动。该提案的创新之处在于开发了一种有针对性的方法来直接量化 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 染色剂的高灵敏度检测。全面的， 该平台将提供第一个定量、低成本且可扩展的方法来利用 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，我们强调该平台的开发 研究转录因子 (TF) 和染色质读取蛋白，尽管我们的平台将广泛适用 用于任何 CAP 的研究。在第一阶段，我们开发了自动化 QuantiCAP 工作流程，证明了可行性 用于量化药物治疗后 CAP 变化的测定，并确定测定条件以进一步 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) 个高价值目标 使用手动和自动工作流程，包括基因组学验证。我们将验证我们的分析 不同的初级免疫细胞和刺激，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，我们将通过开发来为营销和商业化做好准备 QuantiCAP beta 试剂盒并应用自动化测定来进行高影响力的药物开发研究。