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

总结 炎症性肠病（IBD）、克罗恩病（CD）和溃疡性结肠炎（UC）是慢性的 胃肠道（GI）状况。CD可发生在胃肠道沿着的任何地方， 异质性临床表现/结局，具有挑战性的治疗。此外，预测CD的可靠标记物 不存在病程和/或治疗反应。我们（和其他人）已经表明，CD亚型显示出独特的 与结果相关的基因表达谱;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 染色质相关蛋白（ChAP），其调节染色质可及性和基因表达。我们 提出，高分辨率分析注释机械不同的染色质特征可能会解开 CD（和其他复杂疾病）内的异质性，揭示了新的预后生物标志物/治疗靶点。 然而，现有的染色质作图测定法（例如ChIP-seq）由于其不稳定性而不适合于临床研究。 有限的生产量、过高的成本和差的再现性，以及缺乏确定的定量控制。 对于这个快速通道的建议，EpiCypher是与博士合作。 胃肠道生物学和疾病（CGIBD）开发HT-CUTANA™，一种高通量，低成本的基因组 用于下一代临床研究的映射解决方案。该项目的创新之处在于开发了一种96- 孔板CUT&RUN平台（HT-CUTANA）专门针对库存人体组织进行了优化， 节省成本，提高灵敏度和通量，这是使用ChIP-seq不可能实现的。这些试验将 配备EpiCypher专有的spike-in技术，用于定量交叉样本比较， 用于简化数据分析的用户友好型生物信息学工具。EpiCypher已经生成了初步的密钥 CUT&RUN分析自动化的数据，支持该提案的可行性，并吸引了大量的早期兴趣 我们的工业和制药合作伙伴。最终的端到端HT-CUTANA系统将能够完全处理 与<5 days while also delivering >ChIP-seq相比，192个样本节省10倍成本。通过与博士的合作。 Sheikh，我们会将HT-CUTANA应用于一个精致的医生策划的CD和对照患者样本库， 证明了HT-CUTANA在研究CD发病机制基础上的新调节机制中的实用性。在 第一阶段（目标1），我们将开发标准化的96孔板HT-CUTANA方法，用于分析库存的人 结肠组织，目标是从单个库存样品中定位六个靶标，并使用该测定来 区分CD与对照。在II期（目标2）中，我们将建立稳健的自动化HT-CUTANA检测试剂盒， 生物信息学工具，通过提高规模和效率降低分析成本。在目标3中，我们将开发HT- CUTANA套件和服务，并与Sheikh博士合作，将这些工具应用于规模化的定量临床研究 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 并引领重大创新，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.