Rakesh Naduvile Veedu

Background: Hepatic fibrosis is a common response to hepatocyte damage presenting a major global health challenge. Despite its prevalence, there are currently no clinically approved therapeutics. In a previous biomarker discovery, we identified dysregulated expression of microRNAs, particularly miR-25-3p (miR-25), in patients with cystic fibrosis, with or without associated liver disease. Further investigation revealed miR-25 acting as a negative regulator of cross-talk between Notch-1 and TGFβR1 in hepatic stellate cells (HSCs). Targeting key activators of Notch 1 signalling, ADAM-17 and FKBP14, miR-25 suppresses fibrillar collagen expression. Building on these findings, we designed a novel, chemically modified miR-25 mimic (miR-25-C3) with significantly enhanced anti-fibrotic activity compared to commercially available mimics. In this study, we evaluate the therapeutic efficacy of this proprietary miR-25-C3.

Methods: To assess miR-25-C3 efficacy, the human HSC line LX-2 was transfected, confirming downregulation of Notch1 activators and fibrillar collagens. A vitamin A-coupled lipid nanoparticle (VA-lipo) system was then optimized for targeted in vivo delivery to hepatic stellate cells. Cirrhosis was induced in C57Bl6J mice via thioacetamide (TAA; 300 mg/L in drinking water) over 8 weeks. Mice received tail vein injections of VA-lipo–miR-25C3 (0.75 mg/kg) every 3 days while continuing TAA exposure. Two treatment cohorts were established: a 2-week group (5 injections) and a 4-week group (10 injections). Livers were harvested for histopathological evaluation, METAVIR scoring, and qPCR analysis of target genes.

Results: miR-25-C3 mimic showed superior downregulation of Notch 1 activator genes ADAM-17 and FKBP14, compared to a commercially available miR-25 mimic, leading to significant inhibition of TGFβRI and fibrillar collagen expression. Additionally, vitamin A-coupled liposomes effectively targeted miR-25-C3 to HSCs in vivo, ensuring enhanced delivery to the liver (vs off-target organs) and minimizing off-target effects. In the TAA-induced liver fibrosis model, both males and females treated with VA-lipo-miR-25-C3 showed significant downregulation of collagen I, II, III, TGFβRI, ADAM-17, and FKBP14 gene expression, as a direct benefit of miR-25-C3 anti-fibrotic therapy. Notably, only male mice showed regression of fibrillar collagens at the protein level, as evidenced by reduced Sirius Red histochemistry and a significant decrease in METAVIR fibrosis scores.

Conclusion: These findings highlight the potential of miR-25-C3 as a novel and promising anti-fibrotic therapeutic. The use of vitamin A-coupled liposomes ensures the effective targeting of miR-25-C3 to HSCs in the liver. Future studies will focus on the underlying mechanisms driving the sex differences and further investigate the translational potential of miR-25-C3 in preclinical for both males and females.