Myotonic dystrophy type 1 (DM1), the most common adult-onset muscular dystrophy, is caused by toxic CUG repeat expansions in the DMPK transcript that disrupt post-transcriptional gene regulation. Despite major advances in therapeutic development, DM1 still lacks approved disease-modifying treatments. Recent studies identified Musashi-2 (MSI2) as a pathogenic regulator of skeletal muscle dysfunction in DM1, suggesting that its inhibition may represent a complementary therapeutic strategy beyond direct targeting of the DMPK-MBNL axis. Here, we developed and characterized antisense oligonucleotides (ASOs) targeting MSI2 through an integrated in silico, in vitro and in vivo screening pipeline. Screening of rationally designed candidates identified GP-89 as a lead molecule with potent MSI2 silencing activity and a favorable tolerability profile. In human DM1-derived transdifferentiated and primary muscle models, GP-89 reduced MSI2 abundance, improved myogenic differentiation and partially restored disease-associated transcriptional alterations. Transcriptomic analyses revealed recovery of dysregulated pathways related to cytoskeletal organization, intracellular transport and cellular signaling, including a subset of genes associated with MSI2 regulatory networks. In vivo, unconjugated GP-89 showed robust hepatic target engagement but limited skeletal muscle activity. An optimized dosing regimen using oleic acid (OA)-conjugated GP-89 achieved sustained MSI2 suppression across multiple skeletal muscles and peripheral tissues, with activity maintained for up to 45 days after the final administration and no evidence of overt systemic toxicity under the conditions tested. These findings establish MSI2 as a druggable therapeutic target in DM1 and provide the first pharmacological evidence that its sustained inhibition can improve disease-relevant molecular and cellular phenotypes