Enhanced muscle uptake of chemically optimized miR-23b antisense oligonucleotides as lead compounds for Myotonic Dystrophy type 1

We developed chemically optimized antisense oligonucleotides (antimiRs) targeting miR-23b, a microRNA overexpressed in myotonic dystrophy type 1 (DM1), a multisystemic genetic disorder caused by CTG repeat expansions in the DMPK gene. This pathological expansion triggers an RNA sequestration mechanism, where mutant transcripts form ribonuclear foci that bind and deplete MBNL. Combined with miR-23b-mediated repression, these events exacerbate splicing defects and downstream cellular dysfunction. Our approach aimed to restore functional MBNL1 protein levels by antagonizing miR-23b effects. Using a multi-step screening process, we evaluated antimiRs with varied sequences, lengths, chemical modifications, and lipid conjugations. A critical optimization was the inclusion of a 3’-end oleic acid conjugation, together with a specific chemical pattern of modifications, which significantly enhanced the muscle uptake and efficacy of lead candidates, demonstrated robust biological activity in preclinical models, including HSA ^LR and DMSXL mice, and human primary myoblasts. Lead candidates significantly increased MBNL1 protein levels, corrected mis-splicing events, improved functional outcomes such as muscle strength and reduced myotonia, and exhibited efficient in vivo biodistribution to skeletal muscle, addressing a key target tissue of DM1 pathology. Toxicological assessments in vitro revealed a favorable safety profile with minimal immune activation or renal toxicity at preclinical doses. Conservation of the mechanism of action for antimiR inhibition was confirmed in rat and pig fibroblasts. Together, these findings established two lead antimiRs as promising therapeutic candidates for DM1, with improved pharmacokinetic properties, tissue targeting, and safety, and highlight the potential of miRNA-based therapies to address critical molecular defects in the genetic disorder, DM1.