Single nuclei sequencing to understand mechanism of action of the melanocortin 1 receptor agonist Pl8177 in Dextran sodium sulfate (DSS)-induced colitis in rats

PL8177 is a potent and selective agonist of the melanocortin 1 receptor (MC1R). PL8177 has shown efficacy in reversing intestinal inflammation in a cannulated rat ulcerative colitis model. To facilitate oral delivery, a novel, polymer-encapsulated formulation of PL8177 was developed and tested in dextran sulfate sodium induced rat ulcerative colitis model. Rats treated with 50 µg oral PL8177 demonstrated significantly lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle without active drug. We used single nuclei RNA sequencing of colon tissues to characterize the mechanism of action and identify relative cell population and key gene expression changes between treated, healthy and vehicle. Nuclei were isolated from 9 flash-frozen rat colon tissue samples (n=3 for each treatment group: PL8177-50ug, Sham and placebo) and were used to construct 3’ single cell gene expression libraries (Next GEM v3.1) using the 10x Genomics Chromium system (10x Genomics, Pleasanton, CA). Libraries were sequenced with ~150 million read pairs (PE150) per sample on an Illumina NovaSeq sequencing system (Illumina, San Diego, CA). After sequencing, gene counts were generated by Cell Ranger v6.0.1 (10x Genomics) using the rat reference genome Rattus norvegicus 6.0.97. Introns were included in the analysis. CellBender was applied to eliminate empty droplets and technical artifacts. Only genes detected in a minimum of 3 cells, cells with at least 200 genes, and cells with <5% of mitochondrial reads were retained