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

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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.