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: 10240732
• 负责人: Michael J Rosen
• 金额: --
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 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 2021-08-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240732
• 关键词: 3-Dimensional; Address; Adult; Algorithms; American; Bioinformatics; Butyrates; Carbocyanines; Cations; Cell Differentiation process; Cell Respiration; Cells; Child; Chromatin; Chronic; Clinical; Clinical Research; Colon; Complex; Crohn' s disease; Cystic Fibrosis; DNA; Data; Defect; Development; Diet; Digestive System Disorders; Disease; Disease model; Dyes; Elements; Endoscopy; Enhancers; Environment; Epigenetic Process; Epithelial; Epithelial Cells; Exhibits; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genus Hippocampus; Histone Acetylation; Homeostasis; Hospitals; Human; Immune; Impairment; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Intestines; Knowledge; Large Intestine; Lead; Measurement; Medical center; Membrane Potentials; Mendelian disorder; Methods; Microbe; Mitochondria; Modeling; Modification; Molecular Genetics; Mucous Membrane; Nutrient; Organoids; Outcome; Oxidative Stress; Oxygen Consumption; Pathogenesis; Patients; Pediatric ulcerative colitis; Pharmacotherapy; Physiological; Play; Pre-Clinical Model; Process; Regulator Genes; Reporting; Research Personnel; Risk; Role; Science; Single-Gene Defect; Structure; Technology; Testing; Therapeutic; Tissues; Translational Research; Ulcerative Colitis; United States National Institutes of Health; Variant; cell type; disabling symptom; drug development; drug response prediction; drug testing; epigenomics; extracellular; follow-up; functional disability; gene function; healing; histone methylation; human model; human subject; improved; insight; intestinal crypt; intestinal epithelium; microbial; microbiome; mitochondrial dysfunction; mitochondrial membrane; novel; novel therapeutics; nuclease; oxidation; pediatric patients; persistent symptom; pre-clinical; predicting response; predictive modeling; promoter; research study; response; stem cells; three dimensional structure; transcriptome; transcriptome sequencing; transcriptomics; uptake

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 患有溃疡性结肠炎（UC）的儿童和成人，炎症性肠病（IBD）的一种形式， 结肠慢性炎症导致的衰弱症状。生理、分子和遗传观察 所有这些都表明肠上皮功能受损是UC多因素发病机制中的关键因素。 粘膜愈合是一个需要许多协调的上皮功能的过程，是阳性的最佳预测因子。 UC的长期结果。然而，没有可用的治疗方法直接改善结肠上皮细胞， 功能我们的研究小组最近报道了上皮细胞线粒体基因和功能的严重抑制 未接受过治疗的儿童UC患者。上皮细胞定向治疗逆转 线粒体功能障碍现在必须是一个优先事项，但疾病UC的相关验证的临床前模型 缺乏上皮细胞。人类肠道类器官是初级的三维上皮结构 从培养的肠隐窝干细胞分化，其含有肠上皮细胞的所有分化细胞类型。 上皮患者来源的结肠上皮类器官（类结肠）有望成为人类临床前模型，用于 UC药物开发，但它们表现出疾病相关特征的程度尚不清楚。类结肠菌 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，在这方面， 基因只能解释UC的少量风险，因为风险在很大程度上受环境影响。的 环境，包括饮食和微生物组，以及UC中长期炎症的死亡可能会影响 以DNA和染色质的表观遗传修饰的形式进入上皮的可传递变化。我们 假设疾病相关上皮表观基因组和转录组改变以及线粒体 在来自UC患者的类结肠中持续存在功能损害。为了解决这个假设，在目标1中，我们 将使用核酸酶（CUT&RUN）测序和RNA- 在配对的患者原代结肠上皮细胞和类结肠（来源于这些细胞）中进行测序，以表征 儿童UC中疾病相关的上皮表观基因组和转录组改变，并确定是否 这些改变在患者来源的类结肠中持续存在。在目标2中，我们将确定UC线粒体是否 功能障碍反映在患者源性结肠样病变中，并持续存在于粘膜愈合中， 线粒体膜电位和动态氧消耗和丁酸氧化的测量 Seahorse技术我们还将评估细菌代谢产物丁酸盐，一种主要的营养物质， 结肠上皮细胞限制了线粒体功能障碍。这项研究有可能建立病人- 衍生的类结肠作为人类UC疾病模型，与疾病具有功能和表观遗传相似性， 上皮这一发现将从根本上改变我们开发和测试上皮细胞的方法- 针对UC的定向治疗。