(337ag) Sting-Pathway Inhibition to Treat Sting-Associated Inflammatory Conditions

Currently, Lucinda is a student in Dr. John Wilson’s Immunoengineering lab at Vanderbilt University, and she is a PhD candidate in Chemical and Biomolecular Engineering. Her broad interests include biomaterials, nanotechnology, and the delivery of immunotherapeutics. Lucinda has been a co-author on multiple cancer immunotherapy and nucleic acid delivery papers, but her thesis research focuses on the inhibition of the delivery of small molecule STING pathway inhibitors to ameliorate STING-driven inflammatory diseases.

Problem

The cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway is implicated in the development and progression of a myriad of inflammatory diseases including colitis, nonalcoholic steatohepatitis, sepsis, age-related macular degeneration, and cellular senescence. Thus, STING pathway inhibitors (in isolation or in combination with other therapies) could have wide therapeutic application in inflammatory conditions. The cGAS inhibitor RU.521 and the STING inhibitor H-151 have improved outcomes in mouse models of colitis, AKI, and ALS; however, these studies required frequent high-dose i.p. injections of the inhibitors, which limits translatability. Furthermore, long-term use of systemically administered cGAS or STING inhibitors may leave patients vulnerable to viral infections and tumor development. Thus, targeted inhibition of the cGAS/STING pathway may be an attractive, broadly applicable treatment for a variety of cGAS/STING-driven ailments. We hypothesize that targeted, PLGA nanoparticle-mediated delivery of RU.521 and H-151 to tissue-resident macrophages can enhance and sustain inhibition of cGAS/STING signaling in the inflamed tissue to slow or reverse progression of cGAS/STING-driven inflammatory conditions, while increasing the translatability of cGAS/STING inhibitors as therapeutics by decreasing the time between dosing and improving safety.

Methods

RU.521 and H-151 were encapsulated in hydrolytic or ROS-responsive 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 at 37°C PBS was monitored over 7 days. 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. Flow cytometry was utilized to observe changes in M1 and M2-associated surface markers after cGAS/STING activation or inhibition. 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.

Results

NPs were fabricated by o/w emulsion, and the inclusion of an excipient polymer that engages in pi-pi interactions with the inhibitors improved drug loading by up to >7-fold and extended in vitro drug release. All nanoparticle formulations potently blocked cGAS/STING-driven inflammatory responses as well or better 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. 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 both human and 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. NPs reached their target tissues, and artificial models of liver interferonopathy and colitis were established. Finally, the inhibitors decreased cGAS/STING signaling in vivo. Chronic, aberrant overactivation of the cGAS/STING has been implicated in a variety of inflammatory conditions, and the targeted delivery and sustained release of STING pathway inhibitors may help resolve inflammation and improve patient outcomes in STING-associated autoimmune and inflammatory diseases.