PROJECT SUMMARY Crohn’s disease (CD) is characterized by progressive, transmural inflammation that can affect any region of the gastrointestinal tract. Penetrating complications, most often perianal fistulae, will affect up to 50% of CD patients over their disease course. Perianal fistulae are a high morbidity CD complication with poor treatment response. African ancestry (AA) patients have higher rates of perianal disease, including perianal fistula, than European ancestry (EA) CD patients. Many of the genetic advances of the genome wide association era are from studies wherein AA patients are underrepresented. PTGER4 is the only risk locus with strong association signals in AA populations. There is a need to include AA patients in genetic and genomic studies of CD, particularly in the context of perianal fistula, to better understand drivers of disease and improve clinical outcomes. Aim 1 seeks to construct a single cell genomics cohort of AA and EA patients with a history of perianal fistulizing CD. To date, there is no single cell sequencing dataset of CD samples with a focus on the rectum or fistula, nor a dataset comparing single cell gene expression between these ancestry groups in CD. We will perform CITE- sequencing on resection tissues from proctectomies and colectomies in patients with severe CD and history of perianal fistula. We will also collect rectal biopsies from areas of active inflammation and from un-involved rectum or colon for single cell RNA sequencing. From these data, we will identify differential gene expression modules that will be validated by projection onto available bulk RNA sequencing data from larger CD patient cohorts. Single cell capture of both RNA and protein expression will provide unprecedented granularity into cell populations important in fistula progression and illuminate any differences between patient populations. In Aim 2, we will further explore drivers of fistula pathogenesis. We identify IL-17 and PGE2 signaling as candidate pathways that influence epithelial integrity in the context of inflammation and epithelial-mesenchymal transition (EMT), promoting fistula inception. We will culture epithelial monolayers long-term from patient-derived organoids to model epithelial differentiation, barrier function, and EMT. We will use this system to assess the effects of PGE2 and IL-17 receptor agonism and inhibition on epithelial cells, and how this may influence wound healing and mediate fistula inception. In this way, we will be able to interrogate specific signaling pathways and their contributions to epithelial health using functional readouts. The academic environment at Mount Sinai is unparalleled to perform this work. Mount Sinai is home to pioneers in IBD clinical care and research, who collaborate in meaningful ways while working with one of the most diverse patient populations in the world. The training plan for this fellowship takes full advantage. Together, the studies described herein will provid...