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

Jessica J King; Kai Chen; Cameron W Evans; Marck Norret; Ruba Almasri; Nathan J Pavlos; Henry Yl Hui; Qiongxiang Lin; Uditi Bhatt; Stephen G Young; Nicole M Smith; Mehran Nikan; Clive A Prestidge; Haibo Jiang; K Swaminathan Iyer

doi:10.1039/d3sc06773d

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

Chem Sci. 2024 Aug 28;15(38):15690-15697. doi: 10.1039/d3sc06773d. Online ahead of print.

Authors

Jessica J King^{1

2}, Kai Chen^{1

2}, Cameron W Evans^{1

2}, Marck Norret^{1

2}, Ruba Almasri^{1

2

3}, Nathan J Pavlos⁴, Henry Yl Hui⁵, Qiongxiang Lin^{1

2}, Uditi Bhatt^{1

2}, Stephen G Young⁶, Nicole M Smith^{1

2}, Mehran Nikan⁷, Clive A Prestidge³, Haibo Jiang^{1

2

8}, K Swaminathan Iyer^{1

2}

Affiliations

¹ School of Molecular Sciences, The University of Western Australia Perth WA 6009 Australia [email protected].
² ARC Training Centre for Next-Generation Biomedical Analysis, The University of Western Australia Perth WA 6009 Australia.
³ UniSA Clinical & Health Sciences, University of South Australia Adelaide SA Australia.
⁴ School of Biomedical Sciences, The University of Western Australia Perth WA 6009 Australia.
⁵ Translational Cancer Pathology Laboratory, School of Biomedical Sciences, The University of Western Australia Perth WA 6009 Australia.
⁶ Department of Medicine, University of California Los Angeles CA 90095 USA.
⁷ Ionis Pharmaceuticals, Inc. Carlsbad CA 92010 USA.
⁸ Department of Chemistry, Faculty of Science, University of Hong Kong Pok Fu Lam Hong Kong [email protected].

Abstract

Antisense oligonucleotides (ASOs) are a well-established therapeutic modality based on RNA interference, but low cellular uptake, limited ability to direct ASO trafficking, and a range of intracellular barriers to successful activity compromise both gene silencing outcomes and clinical translations. Herein, we demonstrate that polymers can increase ASO internalisation via intracellular trafficking pathways that are distinct from lipid-based delivery reagents. For the first time, we spatially define internalisation and dissociation stages in the polymer-mediated cytosolic delivery of ASOs using Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which enables visualisation of ASO localisation at the organelle level. We find that polymer-ASO complexes are imported into cells, from which free ASO enters the cytosol following complex dissociation. This information enables a better understanding of the intracellular trafficking pathways of nucleic acid therapeutics and may be exploited for therapeutic delivery to enhance the effectiveness of nucleic acid therapeutics in the future.