Naked antisense oligonucleotides remain endolysosomally sequestered despite induced membrane damage

Antisense oligonucleotides (ASOs) enter cells efficiently, but the compartment from which productive escape occurs remains uncertain. We used live-cell microscopy, ratiometric pH measurements and 3D focused ion beam scanning electron microscopy (FIB-SEM) in U2OS cells to track a Malat1-targeting ASO from uptake to delivery. The ASO entered by endocytosis and accumulated in late endosomes, endolysosomes and lysosomes, where it induced luminal neutralization without galectin-3 recruitment or limiting-membrane rupture. Under conditions that reduced Malat1-RNA by >90%, quantitative imaging showed that less than 4% of internalized ASOs reached the nucleus. L-leucyl-L-leucine methyl ester (LLOMe)-induced membrane damage released co-internalized dextran but not ASOs, showing that ASOs remain sequestered even in damaged late endocytic compartments. In apilimod-expanded organelles, ASOs concentrated at limiting membranes and intraluminal foci with constrained motion, consistent with association with membrane and luminal structures. Although G3BP1/2 has been proposed to plug damaged endocytic membranes, we detected no recruitment of G3BP1 to endosomes or lysosomes; loss of G3BP1 and G3BP2 increased functional delivery modestly. We therefore propose that productive escape occurs earlier in endocytosis, most likely in early or recycling endosomes, where ASOs would still be unbound within the lumen and where membrane fusion and fission could generate perforations permitting release.