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Published on: June 15, 2018
Naked antisense oligonucleotides remain endolysosomally sequestered despite induced membrane damage
Ewa Sitarska1,2, Anand Saminathan1,2, Gustavo Scanavachi1,2,3
1Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston Children's Hospital, 200 Longwood Ave, Boston, MA 02115, USA.
Biorxiv : the Preprint Server for Biology
|May 25, 2026
Summary
Antisense oligonucleotides (ASOs) efficiently enter cells but escape late endosomes poorly. Productive release likely occurs earlier in endocytosis, possibly in early or recycling endosomes, for effective delivery.
Area of Science:
- Cell Biology
- Molecular Biology
- Drug Delivery
Background:
- Antisense oligonucleotides (ASOs) are promising therapeutics but their intracellular delivery mechanism is not fully understood.
- Efficient cellular uptake does not guarantee therapeutic efficacy due to intracellular trafficking barriers.
Purpose of the Study:
- To investigate the intracellular trafficking and release mechanism of antisense oligonucleotides (ASOs) from endocytic compartments.
- To identify the cellular compartment responsible for productive ASO escape for therapeutic delivery.
Main Methods:
- Live-cell microscopy to track ASO localization.
- Ratiometric pH measurements to assess endosomal acidification.
- 3D focused ion beam scanning electron microscopy (FIB-SEM) for ultrastructural analysis.
- Quantitative imaging to determine ASO nuclear accumulation.
Main Results:
- ASOs accumulate in late endosomes, endolysosomes, and lysosomes, inducing luminal neutralization.
- Less than 4% of internalized ASOs reached the nucleus, even with >90% target RNA reduction.
- ASOs remained sequestered in damaged late endocytic compartments, and G3BP1/2 recruitment was minimal.
- Loss of G3BP1/2 showed only a modest increase in functional delivery.
Conclusions:
- ASOs are inefficiently released from late endocytic compartments.
- Productive ASO escape likely occurs earlier in endocytosis, such as in early or recycling endosomes.
- Early endosomal escape may allow ASOs to leverage membrane dynamics for release into the cytoplasm.
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