Abstract
The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted endocytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA. It is unknown where or when this happens following GLP1-ASO binding to GLP1R and endocytosis. Here, we use correlative nanoscale secondary ion mass spectroscopy (NanoSIMS) and transmission electron microscopy to explore GLP1-ASO subcellular trafficking in GLP1R overexpressing HEK293 cells. We isotopically label both eGLP1 and ASO, which do not affect the eGLP1-ASO conjugate function. We found that the eGLP1 peptide and ASO are not detected at the same level in the same endosomes, within 30 min of GLP1R-HEK293 cell exposure to eGLP1-ASO. When we utilized different linker chemistry to stabilize the GLP1-ASO conjugate, we observed more ASO located with GLP1 compared to cell incubation with the less stable conjugate. Overall, our work suggests that the ASO separates from GLP1 relatively early in the endocytic pathway, and that linker chemistry might impact the GLP1-ASO function.
Highlights
Antisense oligonucleotides (ASOs) are being explored as nucleic acid therapeutics and are taken up by permissive cell lines via endocytosis
We previously described the development of an antisense oligonucleotides (ASOs) with 15× 34 S incorporated in the PS backbone (34 S15 -ASO) [23]
A recent NanoSIMS study detected the cellular uptake of 34 S-labelled ASOs; it did not attempt to measure 34 S-ASO enrichment following ligand-mediated delivery [22]
Summary
Antisense oligonucleotides (ASOs) are being explored as nucleic acid therapeutics and are taken up by permissive cell lines via endocytosis. This can downregulate the Pharmaceutics 2022, 14, 463. Therapeutic gapmer ASOs are typically extensively modified to improve the binding affinity, while reducing cellular toxicity [4–6]. Linkages between nucleotides improves the nuclease resistance and increases binding to serum proteins [7]; the productive cellular uptake of PS-ASOs is still poor in most cell types. Conjugation to targeting ligands is employed to improve the PS-ASO uptake in nonpermissive cell types and/or to promote uptake by disease-relevant cells. N-acetylgalactosamine (GalNAc) has been employed and studied extensively as a ligand for targeting PS-ASOs to hepatocytes via binding to asialoglycoprotein receptors (ASGPRs) [8]. We previously described the use of ligands targeting the glucagon-like peptide
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
