(Poster 160) In Vivo and in Vitro Changes in cGAS-STING Pathway Using cGAS-inhibitor RU.521

Objectives: To determine the effective dose and timing of cGAS-inhibitor RU.521 required to inhibit the cGAS/STING (cyclic-GMP-AMP synthase/stimulator of interferon genes) pathway in vaginal fibroblasts, both in vitro and in vivo.

Methods: To evaluate an in vitro dose curve, fibroblasts isolated from wild type (WT) mouse vagina were treated with vehicle or RU.521 at concentrations 1, 10, 20, and 30 µM for 24 hours. RNA was extracted and cellular senescence markers were assessed with RT-qPCR. For the in vivo time course, 20-week-old WT (n=30) and Fbln5-/- (n=4) mice were randomized to single intravaginal injection of RU.521 (10 mg/kg) or vehicle. Vaginal tissue was harvested from WT animals 1, 2, 3, 5, or 7 days post-injection, while all Fbln5-/- mice were collected at 7 days. Concentration of cGAS/STING second messenger, 2’3’-cyclic-GMP-AMP (2’3’-cGAMP), was determined with ELISA. RT-qPCR assessed senescence markers in WT at 5 and 7 days. Data were analyzed using Mann-Whitney U and Kruskal-Wallis tests with post hoc analysis employing Bonferroni correction.

Results: In vitro, expression levels of all gene targets varied significantly with RU.521 concentration (cGAS, p=.023; MMP-9, p=.021; MMP-12, p=.046; p21, p=.047) (Figure 1). While no statistically significant pairwise comparisons were noted on post hoc testing, the 20 uM dosage appeared to provide maximal suppression of markers, with no additional benefit observed at higher concentrations. All animals tolerated injections without complication. In vivo, intravaginal RU.521 significantly reduced 2’3’-cGAMP levels at 5 days (0.6 vs sham 30.4 pg/mL, p=.019) (Figure 2A). By 7 days, no statistically significant differences in 2’3’-cGAMP were observed with treatment in either WT (sham 3.4 pg/mL, RU.521 2.6 pg/mL) or Fbln5-/- groups (sham 444.0 pg/mL, RU.521 71.2 pg/mL) (p=.392) (Figure 2B). RU.521 reduced gene expression of cGAS (0.67 vs sham 1.54, p=.05) and MMP-9 (0.09 vs sham 0.27, p=.05) at 7 days in WT, which approached but did not reach statistical significance. No such differences were noted at 5 days.

Conclusions: We identify in vitro and in vivo doses of RU.521 capable of mediating the cGAS/STING pathway without toxicity. In vitro concentration 20 uM resulted in downstream changes in the senescence-associated secretory phenotype (SASP) in murine vaginal fibroblasts. Our findings demonstrate intravaginally administered RU.521 10 mg/kg successfully inhibits second messenger 2’3’-cGAMP and suggest downregulation of SASP may subsequently follow. Future studies investigating RU.521 for senescence-related conditions such as pelvic organ prolapse should further evaluate the frequency and duration of therapy and characterize the full profile of molecular changes achieved.

Figure 1. In vitro gene expression of cellular senescence markers in mouse vaginal fibroblasts treated with RU.521 at varied doses for 24 hours.

Figure 2. In vivo concentration of second messenger 2’3’-cGAMP at various timepoints following RU.521 administration in WT (A) and at 7 days post-injection in WT and Fbln5-/- (B). *p < .05.