ESCRT-I complex enhances therapeutic efficacy of ASO drugs by regulating endosomal escape and glucose homeostasis.

Antisense oligonucleotide (ASO) therapeutics silence gene expression through RNase H-mediated mRNA degradation or steric blockade, yet their clinical efficacy is limited by inefficient intracellular trafficking, with <1% of internalized ASOs escaping endosomes to access target RNA. A deeper understanding of the cellular mechanisms governing ASO trafficking and activity is therefore critical. Here, we identify the endosomal sorting complex required for transport-I (ESCRT-I) as a previously unrecognized regulator of ASO pharmacological activity. Using human hepatocyte-derived cell models and FDA-approved ASO drugs inotersen and mipomersen, we demonstrate that suppression of specific ESCRT-I subunits, VPS23 and VPS28, markedly enhances ASO-mediated target silencing, whereas depletion of other subunits has minimal effect. Mechanistically, VPS23 functions as a central ESCRT-I component linking endosomal trafficking to intracellular glucose homeostasis. Loss of VPS23 reduces expression of the glucose transporter GLUT2, lowers intracellular glucose levels, and possibly enables ASO endosomal escape without altering cellular uptake or RNase H1-dependent activity. VPS23 suppression disrupts endosomal morphology, decreases ASO retention in late endosomes, and enhances cytosolic availability of ASOs. Notably, this regulatory effect extends beyond ASOs to small interfering RNA (siRNA) therapeutics, indicating a broader role for ESCRT-I in RNA drug biology. Together, these findings uncover a metabolic-endosomal axis controlling nucleic acid drug efficacy and provide new mechanistic insight into intracellular determinants of RNA-based therapeutics.