STING-Pathway Inhibiting PLGA Nanoparticles to Treat STING-associated Inflammatory Conditions

Friday, October 13, 2023

2:45 PM – 3:00 PM PDT

Location: 325 (Center, Level 3)

Presenting Author(s)

Lucinda Pastora

PhD Candidate
Vanderbilt University
Nashville, Tennessee, United States

Co-Author(s)

PC

Plamen Christov

Vanderbilt University, United States
CP

Christian Palmer

Author
Vanderbilt University, United States

Primary Investigator(s)

JW

John Wilson

Associate Professor
Vanderbilt University
Nashville, Tennessee, United States

Introduction:: 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.

Materials and Methods:: RU.521 and H-151 were encapsulated in PLGA nanoparticles using oil-in-water (o/w) emulsions stabilized with PVA. 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, the free inhibitors and loaded nanoparticles were tested in a model of liver interferonopathy and colitis.

Results, Conclusions, and Discussions:: Results: All NPs fabricated by o/w emulsion are ~160 nm in diameter. Inclusion of an excipient polymer that engages in pi-pi interactions with the inhibitors improved drug loading by up to >7-fold, and it increased the drug release from 3 days to more than 7 days. All nanoparticle formulations tested 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, NPs accumulated in the target tissues, and the inhibitors decreased cGAS/STING signaling. The therapeutic efficacy of our nanoparticle formulations is currently being evaluated in murine models of STING-driven disease and interferonopathy.

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. 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.

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