Microbiome- and\nHost Inflammasome-Targeting Inhibitor\nNanoligomers Are Therapeutic in the Murine Colitis Model

Autoimmune and autoinflammatory diseases account for\nmore than\n80 chronic conditions affecting more than 24 million people in the\nUS. Among these autoinflammatory diseases, noninfectious chronic inflammation\nof the gastrointestinal (GI) tract causes inflammatory bowel diseases\n(IBDs), primarily Crohn’s and ulcerative colitis (UC). IBD\nis a complex disease, and one hypothesis is that these are either\ncaused or worsened by compounds produced by bacteria in the gut. While\ntraditional approaches have focused on pan immunosuppressive techniques\n(e.g., steroids), low remission rates, prolonged illnesses, and an\nincreased frequency of surgical procedures have prompted the search\nfor more targeted and precision therapeutic approaches. IBD is a complex\ndisease resulting from both genetic and environmental factors, but\nseveral recent studies have highlighted the potential pivotal contribution\nof gut microbiota dysbiosis. Gut microbiota are known to modulate\nthe immune status of the gut by producing metabolites that are encoded\nin biosynthetic gene clusters (BGCs) of the bacterial genome. Here,\nwe show a targeted and high-throughput screening of more than 90 biosynthetic\ngenes in 41 gut anaerobes, through downselection using available bioinformatics\ntools, targeted gene manipulation in these genetically intractable\norganisms using the Nanoligomer platform, and identification and synthesis\nof top microbiome targets as a Nanoligomer BGC cocktail (SB_BGC_CK1,\nabbreviated as CK1) as a feasible precision therapeutic approach.\nFurther, we used a host-directed immune target screening to identify\nthe NF-κB and NLRP3 cocktail SB_NI_112 (or NI112 for short)\nas a targeted inflammasome inhibitor. We used these top two microbe-\nand host-targeted Nanoligomer cocktails in acute and chronic dextran\nsulfate sodium (DSS) mouse colitis and in TNF<sup>ΔARE/+</sup> transgenic mice that develop spontaneous Crohn’s like ileitis.\nThe mouse microbiome was humanized to replicate that in human IBD\nthrough antibiotic treatment, followed by mixed fecal gavage from\n10 human donors and spiked with IBD-inducing microbial species. Following\ncolonization, colitis was induced in mice using 1 week of 3% DSS (acute)\nor 6 weeks of 3 rounds of 2.5% DSS induction for a week followed by\n1 week of no DSS (chronic colitis model). Both Nanoligomer cocktails\n(CK1 and NI112) showed a strong reduction in disease severity, significant\nimprovement in disease histopathology, and profound downregulation\nof disease biomarkers in colon tissue, as assessed by multiplexed\nELISA. Further, we used two different formulations of intraperitoneal\ninjections (IP) and Nanoligomer pills in the chronic DSS colitis model.\nAlthough both formulations were highly effective, the oral pill formulation\ndemonstrated a greater reduction in biochemical markers compared to\nIP. A similar therapeutic effect was observed in the TNF<sup>ΔARE/+</sup> model. Overall, these results point to the potential for further\ndevelopment and testing of this inflammasome-targeting host-directed\ntherapy (NI112) and more personalized microbiome cocktails (CK1) for\npatients with recalcitrant IBD.