Quantitative NanoSIMS provides subcellular concentration and distribution of oligonucleotide therapeutics
Abstract
Antisense oligonucleotides (ASOs) represent a powerful therapeutic modality that can selectively modulate gene expression. However, ASOs face two major hurdles that restrict their use in the clinic. The first issue is delivery of the ASO to a tissue of therapeutic interest while reducing exposure to unrelated tissues. Additionally, inefficient escape of ASOs from endolysosomal compartments affects their activity since ASOs are unable to reach their intracellular RNA target in the nucleus and/or cytosol. Despites the variety of chemical modifications developed to tackle these delivery issues, it remains challenging to reach particular tissues and/or cell types outside of the liver, and there are still no non-toxic solutions to the endosomal escape problem.
To fully realize the therapeutic potential of this class of molecules, it is crucial to understand the mechanisms underlying how ASOs enter cells and exit the endosomal space. Therefore, this thesis focuses on the use of nanoscale secondary ion mass spectrometry (NanoSIMS), in combination with electron microscopy, to investigate the subcellular distribution and accumulation of ASOs.
It was necessary to develop a NanoSIMS method capable of absolute quantification of the intracellular exposure of ASO. Thus, external standards were developed to quantify several halogenated compounds (iodine, bromine, and fluorine) as well as a sulfur isotope (34S).
Results showed that the uptake of different ASOs was saturable, but conjugation to a N-acetylgalactosamine targeting domain enhanced cellular uptake and improved target knockdown. NanoSIMS data also showed that upon colchicine treatment, the uptake and localization of ASOs were affected. It was also possible to quantifying both the targeting domain and ASO components of an engineered glucagon-like peptide 1 ASO conjugate. That highlighted that fine tuning of ASO chemistry can be used to affect the productive uptake of ASOs.
Overall, these findings contribute to a better understanding of the cellular delivery, uptake and trafficking mechanisms of ASOs, which is valuable for the future development of more effective oligonucleotide-based therapeutics.
Parts of work
Paper I: Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS. Cécile Becquart, Rouven Stulz, Aurélien Thomen, Maryam Dost, Neda Najafinobar, Anders Dahlén, Shalini Andersson, Andrew G. Ewing, Michael E. Kurczy. Analytical Chemistry, 2022, 94(29), 10549-10556. https://doi.org/10.1021/acs.analchem.2c02111 Paper II: Nanoscale secondary ion mass spectrometry quantification of targeted drug delivery of nucleic acid-based therapeutics: The GalNAc benchmark. Cécile Becquart, Quentin Vicentini, Rouven Stulz, Anders Dahlén, Samir El Andaloussi, Constanze Hilgendorf, Shalini Andersson, Andrew G. Ewing, Michael E. Kurczy. Under review at ACS Nano 2023 Paper III: Understanding antisense oligonucleotide trafficking with NanoSIMS: quantification of the colchicine effect. Cécile Becquart, Dennis Hekman, Constanze Hilgendorf, Shalini Andersson, Andrew G. Ewing, Michael E. Kurczy. Manuscript. Paper IV: NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution. Emma Kay, Rouven Stulz, Cécile Becquart, Jelena Lovric, Carolina, Tängemo, Aurélien Thomen, Dženita Baždarević, Neda Najafinobar, Anders Dahlén, Anna Pielach, Julia Fernandez-Rodriguez, Roger Strömberg, Carina Ämmälä, Shalini Andersson, Michael E. Kurczy. Pharmaceutics, 2022, 14(2), 463-481. https://doi.org/10.3390/pharmaceutics14020463
Degree
Doctor of Philosophy
University
University of Gothenburg. Faculty of Science.
Institution
Department of Chemistry and Molecular Biology ; Institutionen för kemi och molekylärbiologi
Disputation
Torsdagen den 15 juni 2023, kl. 10:00, i sal 10:an, Kemigården 4 (Chalmersområdet), Göteborg
Date of defence
2023-06-15
cecile.becquart@gu.se
Date
2023-04-28Author
Becquart, Cécile
Keywords
NanoSIMS
Antisense Oligonucleotide
Intracellular concentration
Publication type
Doctoral thesis
Language
eng