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

项目总结 儿童和成人溃疡性结肠炎(UC)是炎症性肠病(IBD)的一种，患有 慢性结肠炎引起的衰弱症状。生理学、分子和遗传学观察 所有这些都表明肠上皮功能受损是UC多因素发病的关键因素。 粘膜愈合是一个需要许多协调的上皮功能的过程，是阳性的最佳预测指标。 UC的长期结果。然而，目前还没有可以直接改善结肠上皮的治疗方法。 功能。我们小组最近报道了上皮细胞线粒体基因和功能的严重抑制。 治疗--幼稚的儿童UC患者。上皮导向治疗逆转的研究进展 线粒体功能障碍现在必须是优先事项，但相关的已验证的UC临床前模型 上皮细胞缺乏。人体肠道器官是原生的三维上皮结构 在培养中从肠道隐窝干细胞分化而来，该干细胞包含所有已分化的肠道细胞类型 上皮组织。患者来源的结肠上皮器物(结肠样体)有望成为人类临床前模型 UC药物开发，但它们在多大程度上表现出与疾病相关的特征尚不清楚。类殖民地 已经成功地用于预测由单基因缺陷引起的疾病的药物反应。然而， 基因只能解释UC的一小部分风险，因为风险在很大程度上受到环境的影响。这个 环境，包括饮食和微生物群，以及UC长期炎症的费用可能会渗透到 以DNA和染色质的表观遗传修饰的形式传递到上皮细胞的变化。我们 假设疾病相关的上皮细胞表观基因组和转录改变与线粒体 UC患者的结肠样体持续存在功能损害。为了解决这一假设，在目标1中，我们 将在靶标下进行平行切割，并使用核酸酶(切割和运行)测序和RNA- 配对患者原代结肠上皮细胞和结肠样物质(来源于这些细胞)中的SEQ特征 儿童UC的疾病相关上皮表观基因组和转录改变，并确定是否 这些变化在患者来源的结肠样体中持续存在。在目标2中，我们将确定UC线粒体 功能障碍反映在患者衍生的结肠样体中，并通过黏膜愈合持续存在 线粒体膜电位、动态耗氧量和丁酸氧化的测定 使用海马体技术。我们还将评估细菌代谢物丁酸盐，一种主要的营养物质 结肠上皮细胞，限制这种线粒体功能障碍。这项研究有可能确定患者- 作为一种功能和表观遗传学与患者相似的人类溃疡性结肠病模型 上皮组织。这样的发现将从根本上改变我们开发和测试上皮细胞的方法- UC的定向治疗。

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.