Abstract Background Intestinal fibrosis remains a major unmet need in Crohn disease, driving stricturing complications which lack effective antifibrotic therapies1. Gremlin-1 (GREM1), a BMP antagonist strongly induced by profibrotic cues, has emerged as a potential master regulator of fibroblast activation2. We aimed to validate GREM1 as a therapeutic target in primary human small-intestinal fibroblasts (HSIFs) and to develop a siRNA-APE nanoparticle (NP) platform for targeted GREM1 silencing. Methods HSIFs were isolated from commercially sourced ileal resections and cultured on extracellular matrix-based gel. Fibroblasts were stimulated with TGFβ1 and/or pro-inflammatory cytokines (IL-17, TNFα) to induce fibrogenic or inflammatory activation (n = 6). A library of 16 APE-NPs encapsulating siGREM1 was screened for viability, transfection efficiency, and knockdown potency. Lead candidates were characterized for size, charge, siRNA encapsulation, and dose response. Antifibrotic activity was assessed by qPCR, ELISA, Western blot, and analysis of phospho-SMAD2/3 signaling activation, with scramble-NP and naked siRNA as controls. Results Several APE-NP formulations enabled efficient siRNA delivery while maintaining 95% viability. The optimized NP showed uniform size, high siRNA loading, and strong transfection. In TGFβ-activated HSIFs, NP-siGREM1 achieved 85-92% GREM1 knockdown at transcript and protein levels. GREM1 silencing induced broad antifibrotic reprogramming, significantly reducing CTGF, COL1A1, and α-SMA (all p 0.05 vs scramble). Notably, NP-mediated GREM1 knockdown completely abolished SMAD2/3 phosphorylation, indicating full blockade of canonical TGFβ signaling. Responses were consistent across independent experiments and markedly superior to scramble-NP and naked siRNA. Conclusion Targeted GREM1 silencing using an optimized siRNA-APE-NP platform reverses fibroblast activation and fully suppresses TGFβ signaling, identifying GREM1 as a central regulator of intestinal fibrosis. These findings support the development of GREM1-directed RNA therapeutics as a promising antifibrotic strategy for Crohn disease to be confirmed by In vivo evaluation and mucosal delivery. References: 1. Amamou A, Leboutte M, Breton J, et al. Mineralocorticoid receptor activation contributes to intestinal fibrosis through neutrophil gelatinase-associated lipocalin in preclinical models. Nat Commun. 2025;16(1):6318. doi:10.1038/s41467-025-61401-0 2. Acharjee A, Shivaji U, Santacroce G, et al. Novel Transcriptomic Signatures in Fibrostenotic Crohn’s Disease: Dysregulated Pathways, Promising Biomarkers, and Putative Therapeutic Targets. Inflamm Bowel Dis. 2025;31(6):1502-1513. doi:10.1093/ibd/izaf021 Conflict of interest: Amamou, Asma: No conflict of interest Deery, Alan: No conflict of interest Lopez Espinar, Aida: No conflict of interest O’Mahony, Cian: No conflict of interest Toman, Jan: No conflict of interest Iacucci, Marietta: Grant: Pentax, Olympus, Eli lilly, Helmsley Personal Fees: Pentax, Pfitzer, Janssen, EliLilly, J & J Kowalski, Piotr S.: No conflict of interest Ghosh, Subrata: None None None
Amamou et al. (Thu,) studied this question.