(265f) ROS-Responsive, on-Demand Delivery of Sting Pathway Inhibitors to Treat Ulcerative Colitis and Reprogram the Immune Microenvironment in the Colon

Problem: Overactivation of the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway has been implicated in the development and progression of ulcerative colitis (UC). STING expression is increased in the colons of both UC patients and mice with experimentally-induced colitis. Furthermore, accumulation of STING in macrophages and monocytes was recently shown to be a driving factor in intestinal inflammation, possibly via Th1 T cell recruitment. Thus, the cGAS/STING pathway is a promising and largely unexplored therapeutic target to treat intestinal inflammation. Small molecule inhibitors of cGAS and STING have recently been developed, and preliminary data from our group, and others, has demonstrated that pharmacological inhibition of cGAS/STING can improve outcomes in murine models of colitis. However, these agents are administered systemically via intraperitoneal injection, limiting potential for translation. Moreover, chronic systemic inhibition of cGAS/STING may leave patients vulnerable to viral infections and tumor development. Our overall objective is to address these major translational barriers through the oral delivery of cGAS and STING inhibitors in reactive oxygen species (ROS)-scavenging nanoparticles that will enhance and sustain inhibition of cGAS/STING signaling in the inflamed colon. We hypothesize that this novel oral nanoparticle delivery system will reverse colitis progression while eliminating potential adverse immunosuppressive systemic effects.

Methods: cGAS and STING inhibitors were encapsulated in ROS-responsive PPS nanoparticles. The size of these particles was measured using dynamic light scattering, drug loading and encapsulation efficiency were quantified using high performance liquid chromatography, and in vitro drug release was monitored over biologically relevant timescales. Type-I interferon reporter cell assays were used to assess the capacity of the inhibitors and particle formulations to suppress cGAS/STING signaling in RAW-Dual cells and THP1-Dual cells stimulated with the cGAS agonist herring testes (HT) DNA. RT-qPCR and Western blots were used to assess cGAS/STING pathway activation in primary macrophages treated with a cGAS agonist and inhibitor. Additionally, a DSS-induced colitis model was used to determine cGAS/STING activation in colitis, and the therapeutic efficacy of free inhibitors and loaded nanoparticles are being tested in our murine model of colitis. Finally, we will use flow cytometry to investigate how cGAS/STING blockade reprograms the immune microenvironment of the lamina propia in the context of colitis.

Results: All NPs fabricated by o/w emulsion are ~160 nm in diameter. Inclusion of an excipient polymer improved drug loading and extended the release profile. All nanoparticle formulations were as potent, if not more potent, in blocking cGAS/STING-driven inflammatory responses than dose-matched free drug in both human monocyte and murine macrophage cell lines and primary macrophages in vitro. Treating M0 BMDMs with inhibitor-loaded NPs decreased M1-like gene expression and cell surface markers when co-administered with a cGAS agonist. In vivo, a DSS-induced murine colitis model was established, and cGAS/STING signaling increased in the diseased colon compared to healthy controls. Systemically administered free drug decreased disease severity, and the therapeutic efficacy of our nanoparticle formulations is currently being evaluated in our colitis model.

Conclusions and Implications: All nanoparticle formulations were as potent or more potent in inhibiting cGAS/STING signaling compared to dose-matched free drug in macrophage cell lines and primary murine macrophages in an in vitro model of interferonopathy. Treating M0 BMDMs with inhibitor decreased M1-like gene expression and cell surface markers when co-administered with a cGAS agonist. Additionally, our preliminary studies demonstrate that inhibition of cGAS and STING can abrogate DSS-induced colitis, and we will assess our new technology for “on-demand” delivery of small molecule cGAS and STING inhibitors in this disease context. This project will advance our knowledge of how cGAS/STING signaling potentiates the development and progression of UC, and will yield a new technology for localized, ROS-responsive blockade of cGAS and/or STING in inflamed intestinal tissue.