High-resolution visualisation of antisense oligonucleotide release from polymers in cells.

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• Antisense oligonucleotides (ASOs) are RNA interference therapeutics with limited cellular uptake and intracellular barriers.
• Current delivery methods face challenges in directing ASO trafficking and achieving effective gene silencing.

Purpose of the Study:

• To investigate polymer-based delivery systems for enhancing ASO cellular internalisation and cytosolic delivery.
• To elucidate the intracellular trafficking pathways of polymer-mediated ASO delivery.

Main Methods:

• Utilisation of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) for spatial and organelle-level visualisation of ASO localisation.
• Development of polymer-ASO complexes for cellular delivery studies.

Main Results:

• Polymers significantly increase ASO internalisation via distinct intracellular pathways compared to lipid-based reagents.
• NanoSIMS imaging revealed polymer-ASO complex import and subsequent dissociation, enabling free ASO release into the cytosol.
• The study provides the first spatial definition of internalisation and dissociation stages in polymer-mediated ASO delivery.

Conclusions:

• Polymer-mediated delivery offers a promising strategy to overcome cellular barriers for ASO therapeutics.
• Understanding these trafficking pathways can guide the development of more effective nucleic acid-based therapies.
• This approach has the potential to enhance the clinical translation and efficacy of ASO treatments.