Studying the subcellular distribution and concentration of antisense oligonucleotides by correlative NanoSIMS and electron microscopy

Wednesday. August 25, 2021

Cécile Becquart^1,2, Rouven Stulz³, Emma Kay³, Jelena Lovric¹, Aurélien Thomen², Kajsa Kanebratt¹, Constanze Hilgendorf¹, Andrew Ewing², Anders Dahlén³, Shalini Andersson³, Michael Kurczy¹.

1 DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden

2 Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden

3 Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden

Abstract

The biodistribution and concentration of antisense oligonucleotides (ASOs) at the subcellular level is proving to be central to the mechanism of action for this class of drugs. The key challenge is to determine if the compound is reaching its intracellular target at sufficient concentration to achieve optimal potency and minimal toxicity. Various techniques have been established to study the intracellular distribution of ASOs. However, those studies usually require the use of labelled probes or compounds, like fluorescent dyes, that may alter the ASO properties and in some cases exaggerate drug toxicity. In parallel, quantitative analysis of ASOs generally depends on biochemical assays and/or LC-MS. But those methods rely mostly on lysate preparations and those cannot easily be used to provide subcellular localization information.

Nanoscale secondary ion mass spectrometry (NanoSIMS) has already proven useful to study the subcellular distribution of a variety of elementally and/or isotopically labeled therapeutics in intact cells. We used NanoSIMS imaging to detect a 34S-labeled GalNAc-MALAT1 conjugate or a bromine-labeled-MALAT1 in liver spheroids at different time points. Additionally, correlation of NanoSIMS with scanning electron microscopy (SEM) allowed the identification of subcellular structures corresponding to ASOs accumulation.

Correlative NanoSIMS and SEM imaging of the 34S-labeled-ASOs-treated hepatocytes reveals that after a 5-min exposure, the 34S-labeled ASO could be detected in multivesicular bodies. The combination of both techniques also shows the accumulation of labeled ASOs within organelles of the endolysosomal pathway for longer incubation periods. Furthermore, the labeled ASOs were only detected in cells from the outer region of the liver spheroids at short exposure times while ASOs could be detected in cells at the center of the spheroids after a 24-h incubation period. Additionally, enrichments of ASOs detected by NanoSIMS for the different experimental conditions were translated into relevant concentrations via the use of homemade standards to follow the evolution of the labeled ASOs signals.