Epigenetic and functional determination of colon organoids as a patient-specific preclinical model of ulcerative colitis

结肠类器官作为溃疡性结肠炎患者特异性临床前模型的表观遗传学和功能测定

• 批准号: 10595943
• 负责人: Michael J Rosen
• 金额: $ 18.75万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10595943
• 关键词: Epigenetic; functional; determination; colon; organoids

PROJECT SUMMARY Children and adults with ulcerative colitis (UC), a form of inflammatory bowel disease (IBD), suffer with debilitating symptoms from chronic inflammation of the colon. Physiologic, molecular, and genetic observations all point to impaired intestinal epithelial function as a key element in the multifactorial pathogenesis of UC. Mucosal healing, a process that requires many coordinated epithelial functions, is the best predictor of positive long term outcomes in UC. However, there are no available treatments that directly improve colon epithelial function. Our group recently reported profound suppression of mitochondrial genes and function in the epithelium of treatment-naive pediatric patients with UC. The development of epithelial-directed treatments to reverse mitochondrial dysfunction must now be a priority, but relevant validated preclinical models of the diseased UC epithelium are lacking. Human intestinal organoids are primary three-dimensional epithelial structures differentiated from intestinal crypt stem cells in culture, which contain all differentiated cell types of the intestinal epithelium. Patient-derived colon epithelial organoids (colonoids) hold promise as a human preclinical model for UC drug development, but the extent to which they exhibit disease-associated features is unknown. Colonoids have been successfully used to predict drug response in diseases caused by a single gene defect. However, genes explain only a small amount of risk for UC, as risk is largely influenced by the environment. The environment, including diet and the microbiome, and the toll of longstanding inflammation in UC likely imbue transmissible changes into the epithelium in the form of epigenetic modifications to DNA and chromatin. We hypothesize that disease-associated epithelial epigenomic and transcriptomic alterations and mitochondrial functional impairment persist in colonoids derived from UC patients. To address this hypothesis, in Aim 1, we will perform parallel Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing and RNA- seq in paired patient primary colon epithelial cells and colonoids (derived from those cells) to characterize disease-associated epithelial epigenomic and transcriptomic alterations in pediatric UC and determine whether these alterations persist in patient-derived colonoids. In Aim 2, we will determine whether UC mitochondrial functional impairment is mirrored in patient-derived colonoids and persists with mucosal healing through measurement of mitochondrial membrane potential and dynamic oxygen consumption and butyrate oxidation with Seahorse technology. We will also assess whether the bacterial metabolite butyrate, a primary nutrient for colon epithelial cells, limits this mitochondrial dysfunction. This study has the potential to establish patient- derived colonoids as a human UC disease model with functional and epigenetic similarities to the diseased epithelium. Such a finding would fundamentally transform our approach to developing and testing epithelial- directed treatments for UC.

项目摘要 儿童和成人溃疡性结肠炎（UC）是一种炎症性肠病（IBD），患有 导致结肠慢性炎症的症状。生理，分子和遗传观察 肠上皮功能损害了UC多因素发病机理的关键元素。 粘膜愈合是需要许多协调上皮功能的过程，是正面的最好的预测指标 UC的长期结果。但是，没有可直接改善结肠上皮的可用疗法 功能。我们的小组最近报道了线粒体基因的深刻抑制和上皮的功能 UC治疗的儿科患者的治疗。上皮指导治疗的发展以逆转 线粒体功能障碍现在必须是优先事项，但相关验证的临床前模型 缺乏上皮。人肠癌是主要的三维上皮结构 培养中与肠道隐窝干细胞区分，其中包含肠道的所有分化细胞​​类型 上皮。患者衍生的结肠上皮器官（结石）作为人类的临床前模型有望 UC药物开发，但是它们表现出与疾病相关的特征的程度尚不清楚。结石 已成功用于预测由单个基因缺陷引起的疾病中的药物反应。然而， 基因仅解释了UC的少量风险，因为风险在很大程度上受环境的影响。这 环境，包括饮食和微生物组，以及UC中长期发炎的损失可能 可传播的变化以表观遗传学修饰的DNA和染色质的形式向上皮变化。我们 假设与疾病相关的上皮表观基因组和转录组改变以及线粒体 从UC患者衍生出的结石型的功能障碍持续存在。为了解决这一假设，在AIM 1中，我们 将在目标下进行平行裂解，并使用核酸酶（切割和运行）测序和RNA-释放 在配对的患者原发性结肠上皮细胞和结石素（源自这些细胞）中的SEQ以表征 小儿UC中与疾病相关的上皮表观基因质和转录组改变，并确定是否是否 这些改变持续存在于患者衍生的结石中。在AIM 2中，我们将确定UC线粒体是否 功能障碍反映在患者来源的结石中，并通过粘膜愈合而持续 线粒体膜电位和动态氧的测量和丁酸酯氧化 使用海马技术。我们还将评估细菌代谢物丁酸酯是否是主要营养素 结肠上皮细胞限制了这种线粒体功能障碍。这项研究有可能建立患者 - 作为人类UC疾病模型的衍生结肠造影，与患病的功能和表观遗传相似 上皮。这样的发现从根本上将改变我们开发和测试上皮的方法 UC的定向治疗方法。