EPHA2/CD44-directed trafficking enhances endosomal leakiness and antisense therapy delivery

The potential for using therapeutic antisense oligonucleotides (ASOs) has been hampered by a lack of understanding of how they enter cells and subsequently access their targets. Endocytosis contributes to ASO uptake, but the machinery mediating subsequent ASO trafficking to permit suppression of their target mRNAs has not been described. Here, we show that direct ASO engagement with a scavenger receptor (CD44) activates the ERK-RSK axis to promote serine phosphorylation of a receptor tyrosine kinase (EPHA2). Serine phosphorylation of EPHA2 permits endocytosis, trafficking, and accumulation of ASOs in nuclear-captured endosomes. These endosomes are then subject to lipid peroxidation and become leaky, allowing ASOs to escape and effectively suppress target mRNA expression. Inhibition of stress granule-mediated repair of these leaky endosomes further enhances ASO effectiveness. These data identify an endocytic route to the nucleus which may be exploited to maximize the effectiveness of ASO-mediated therapies.