Development of a high-throughput epigenomic mapping platform to molecularly phenotype Crohn's disease

摘要 炎症性肠病(IBD)、克罗恩病(CD)和溃疡性结肠炎(UC)是慢性疾病 胃肠道(GI)状况。Cd可发生在胃肠道的任何地方，并有很高的 不同的临床表现/结果，具有挑战性的治疗。此外，预测CD的可靠标记 病程和/或治疗反应不存在。我们(和其他人)已经证明了CD子类型显示为唯一的 基因表达谱与结果相关；然而，潜在的调控机制仍然难以捉摸。 转录受组蛋白翻译后修饰(PTM)和组蛋白翻译后修饰(PTM)的联合作用控制。 染色质相关蛋白(CHAP)，它调节染色质的可及性和基因表达。我们 提出高分辨率分析注释机制上不同的染色质特征可能会解开 CD(和其他复杂疾病)中的异质性，揭示了新的预后生物标志物/治疗靶点。 然而，现有的染色质图谱分析方法(如CHIP-SEQ)由于其局限性而不适用于临床研究。 产量有限、成本过高、再现性差，以及缺乏明确的定量控制。 对于这项快速通道提案，EpiCypher正在与Shehzad Sheikh博士和北卡罗来纳州大学的中心合作 胃肠生物学与疾病研究开发高通量、低成本基因组HT-Cutana™ 下一代临床研究的地图解决方案。该项目的创新之处在于开发了一款96- 井板切割运行平台(HT-Cutana)专门针对储存的人体组织进行优化，提供大量 在灵敏度和吞吐量方面的成本节约和收益是使用芯片序列所不能实现的。这些化验结果将是 配备EpiCypher专有的尖峰技术，用于定量跨样本比较，以及 用户友好的生物信息学工具，用于简化数据分析。EpiCypher已生成初步密钥 用于切割和运行分析自动化的数据，支持此建议的可行性，并吸引了早期的重大兴趣 来自我们的工业和制药合作伙伴。最终端到端的HT-Cutana系统将能够完全处理 192个样品在&lt；5天内交付，同时交付&gt；成本比芯片节省10倍。通过我们与Dr. 酋长，我们将把HT-Cutana应用到医生精心策划的CD库中，并控制患者样本， 证明了HT-Cutana在研究CD发病的新的调控机制方面的作用。在……里面 第一阶段(目标1)，我们将开发标准化的96孔板HT-Cutana方法，用于分析银行人员 结肠组织，目标是从一个库样本中定位六个靶点，并使用这种分析方法来 区分CD和控件。在第二阶段(目标2)，我们将建立强大的自动化HT-Cutana分析和 生物信息学工具，通过提高规模和效率来降低检测成本。在目标3中，我们将开发HT- Cutana工具包和服务，并与Sheikh博士合作，将这些工具应用于规模化、量化的临床研究 在CD患者样本中。这些实验将使HT-Cutana成为生物医学研究的有力工具 并带头进行重大创新，以揭示新的CD机制和预后指标。

SUMMARY The inflammatory bowel diseases (IBDs), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic conditions of the gastrointestinal (GI) tract. CD can occur anywhere along the GI tract and has highly heterogeneous clinical presentation/outcomes, challenging treatment. Further, reliable markers that predict CD course and/or treatment response do not exist. We (and others) have shown that CD subtypes display unique gene expression profiles associated with outcomes; yet, underlying regulatory mechanisms remain elusive. Transcription is controlled by the combined effects of histone post-translational modifications (PTMs) and chromatin associated proteins (ChAPs), which modulate chromatin accessibility and gene expression. We propose that high-resolution assays annotating mechanistically distinct chromatin features may unravel the heterogeneity within CD (and other complex diseases), revealing new prognostic biomarkers/therapeutic targets. However, existing chromatin mapping assays (e.g. ChIP-seq) are unsuitable for clinical studies due to their limited throughput, prohibitive costs, and poor reproducibility, as well as a lack of defined quantitative controls. For this Fast-Track proposal, EpiCypher is partnering with Dr. Shehzad Sheikh and the UNC’s Center for Gastrointestinal Biology and Disease (CGIBD) to develop HT-CUTANA™, a high-throughput, low-cost genomic mapping solution for next-generation clinical research. The innovation of this project is the development of a 96- well plate CUT&RUN platform (HT-CUTANA) specifically optimized for banked human tissues, providing massive cost savings and gains in sensitivity and throughput that are impossible using ChIP-seq. These assays will be equipped with EpiCypher’s proprietary spike-in technologies for quantitative cross-sample comparisons, and user-friendly bioinformatic tools for streamlined data analysis. EpiCypher has already generated key preliminary data for CUT&RUN assay automation, supporting feasibility of this proposal and drawing significant early interest from our partners in industry and pharma. The final end-to-end HT-CUTANA system will be able to fully process 192 samples in <5 days while also delivering >10-fold cost savings vs. ChIP-seq. Via our partnership with Dr. Sheikh, we will apply HT-CUTANA to an exquisite physician-curated bank of CD and control patient samples, demonstrating the utility of HT-CUTANA to study novel regulatory mechanisms underlying CD pathogenesis. In Phase I (Aim 1), we will develop standardized 96-well plate HT-CUTANA methods for analysis of banked human colon tissue, with the goal of mapping six targets from a single banked sample and using this assay to discriminate CD vs. controls. In Phase II (Aim 2), we will establish robust automated HT-CUTANA assays and bioinformatics tools, driving down assay costs by increasing scale and efficiency. In Aim 3, we will develop HT- CUTANA kits and services, and work with Dr. Sheikh to apply these tools for scaled, quantitative clinical research in CD patient samples. These experiments will establish HT-CUTANA as a powerful tool for biomedical research and spearhead major innovations to reveal novel CD mechanisms and prognostic indicators.