Extracellular matrix-targeting oligonucleotide reverses marrow fibrosis

Abstract Myelofibrosis (MF) is a severe hematologic malignancy characterized by clonal stem cell-derived myeloproliferation, splenomegaly, and progressive bone marrow fibrosis, ultimately leading to cytopenias1. While, JAK inhibitors provide meaningful clinical benefits, primarily through spleen volume reduction and symptom relief, their impact on bone marrow fibrosis remains limited2. Fibronectin (FN), a core component of interstitial extracellular matrix (ECM), plays a crucial role in ECM assembly by serving as a scaffold for other matrix proteins and regulating diverse cellular functions. FN and its spliced isoforms are aberrantly expressed in the fibrotic matrix of MF and contribute to disease progression. Elevated expression of EDA containing-FN has been shown to drive megakaryocytic hyperplasia, fibrosis, and inflammation3. Furthermore, increased expression of FN receptors and enhanced FN-binding capacity have been documented in JAK2V617F-mutant hematopoietic progenitors and megakaryocytes4. Despite these insights, effective therapies that directly disrupt ECM remodeling and its contribution to fibrosis remain a significant unmet clinical need in MF. Here, we present a novel antisense oligonucleotide (ASO) designed to suppress the expression of all FN isoforms as a direct anti-fibrotic strategy. We developed a chemically modified 2′-O-(2-methoxyethyl) phosphorothioate ASO targeting a highly conserved sequence shared between human and murine genomes, effectively silencing all cellular FN splice variants. In tissue culture, this ASO induced a sequence- and concentration-dependent reduction in FN mRNAs, including transcripts encoding the profibrotic EDA isoform. In vitro, FN ASO treatment of human bone marrow-derived mesenchymal stromal cells attenuated the acquisition of a myofibroblast phenotype induced by Transforming Growth Factor-β1 (TGF-β1), as indicated by reduced expression of α-smooth muscle actin (α-SMA) and impaired cell migratory capacity. In vivo, systemic subcutaneous administration of FN ASO in C57BL/6 mice (50 mg/kg twice weekly for two weeks) led to a robust reduction of FN mRNA and protein in the liver, along with an approximately 90% decrease in circulating plasma FN levels, as measured by ELISA. Biodistribution studies confirmed efficient targeting of tissue FN, including in the bone marrow and spleen, with approximately 50% reduction in FN protein levels. Importantly, no significant alterations were observed in blood markers of liver or kidney function, indicating a favorable safety profile. In a preclinical Romiplostim-induced mouse model of MF, FN ASO treatment significantly reduced reticulin deposition in the bone marrow, attenuated megakaryocyte expansion, and decreased expression of fibrogenic cytokines such as TGF-β1 and IL-6. While spleen weights remained unchanged, FN ASO-treated mice exhibited marked reversal of reticulin fibers, reduced type III collagen content, and diminished accumulation of α-SMA positive cells, consistent with decreased myofibroblast activity. These findings establish FN ASO as a first-in-class therapeutic approach that directly targets fibrosis in MF, with promising potential for broader application across fibrotic diseases. References D. A. Arber et al., The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 127, 2391-2405 (2016). A. Tefferi, Primary myelofibrosis: 2023 update on diagnosis, risk-stratification, and management. Am J Hematol 98, 801-821 (2023). A. Malara et al., EDA fibronectin-TLR4 axis sustains megakaryocyte expansion and inflammation in bone marrow fibrosis. J Exp Med 216, 587-604 (2019). S. Matsuura et al., Adhesion to fibronectin via α5β1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis. Blood. 135, 2286-2291 (2020).