Preclinical development of a Meg3 ASO inhibitor for the treatment of cardiac fibrosis

Background Cardiac fibrosis (CF), a hallmark of pathological cardiac remodeling, contributes significantly to heart failure (HF) and associated mortality. Characterized by excessive extracellular matrix deposition, CF leads to increased myocardial stiffness and impaired cardiac function. Despite improvements in treatment strategies, the prospects of cure for patients with advanced HF derived from CF remain poor, indicating the urgent need for innovative therapeutic strategies specifically targeting CF. Purpose Long non-coding RNAs (lncRNAs) have emerged as critical regulators in various pathophysiological processes, including cardiovascular disease. The conserved lncRNA Meg3 was found enriched in cardiac fibroblasts and dysregulated in HF models. Moreover, the role of Meg3 and the anti-fibrotic effects caused by its inhibition were validated in mice in preceding studies. Thus, this study aimed to enhance the translational potential of the anti-Meg3 approach by developing an optimized, novel lncRNA-based antisense oligonucleotide therapeutic for HF derived from CF close to clinical readiness. Methods Through large-scale screening, a sequence-optimized GapmeR specific for humans and relevant model species was identified, enabling Meg3 silencing both in vitro and in vivo. In vitro, human cardiac fibroblasts (HCFs) and multicellular human cardiac organoids (hCOs) were stimulated with TGFβ1 to induce fibrosis and assess the anti-fibrotic efficacy of MEG3 inhibition. Mechanistic insights were obtained via bulk RNA sequencing and pulldown assays in HCFs. For preclinical development, pharmacokinetic, toxicological, and pharmacodynamic studies were conducted in rats, minipigs, and domestic pigs. Results GapmeR-mediated MEG3 inhibition led to a significant downregulation of fibrosis-associated markers at both transcript and protein levels in HCFs and hCOs, confirming its anti-fibrotic potential in vitro. These results were validated through transcriptomic profiling after MEG3 inhibition revealing modulation of fibrosis-related signaling pathways. To elucidate the underlying molecular mechanisms, pulldown assays followed by mass spectrometry were performed, uncovering potential MEG3-binding proteins known to participate in fibrotic signaling cascades. In vivo, pre-clinical studies demonstrated efficient Meg3 silencing across rodent and porcine models, with no target-related safety concerns observed. Importantly, pharmacodynamic evaluation in a large animal HF model showed that GapmeR treatment attenuated pathological cardiac remodeling. Conclusions Inhibition of Meg3 induced promising anti-fibrotic effects across in vitro and in vivo models and mitigated cardiac remodelling, which supports the therapeutic relevance of the anti-Meg3-strategy in a translational context. Mechanistic studies provided first insights into the mode of action underlying these protective effects. These findings pave the way for further clinical development.