Evaluation, synthesis and imaging of therapeutic oligonucleotides

A new method for the ³⁴S isotope labelling of phosphorothioate oligonucleotides is reported which allows a practical synthesis of site specifically labeled oligonucleotides. Several applications of this method are reported. It was successfully applied to the quantification of oligonucleotides in tissues and showed superior performance to previously used methods. The application of ³⁴S labeled phosphorothioate oligonucleotides to quantitative Nano Secondary Ion Mass Spectrometry is also shown. Oligonucleotides conjugated to a GLP1 binding peptide were shown to decouple from the peptide quickly in endosomes and escape to the cytoplasm. This process can be influenced by the linker chemistry and helps explain different efficacies in mRNA knockdown when using different conjugation chemistries.

The synthesis and scaleup of 2′-O-AECM nucleotide phosphoramidites is described which includes a new protecting strategy and optimization of reaction conditions. Application of those phosphoramidites in the synthesis of fully 2′-O-AECM modified gapmers is described and they are shown to be active in knocking down MALAT1. 2′-O-AECM modified gapmers are shown to achieve a high level of knockdown if they are conjugated to an endosomal escape enhancing peptide.

List of scientific papers

I. Rouven Stulz, Fiona Milligan, Craig Stovold, Iain Love, Roger Strömberg, Shalini Andersson, and Anders Dahlén. ³⁴S-SIL of PCSK9-Active Oligonucleotide as Tools for Accurate Quantification by Mass Spectrometry. Nucleic acid therapeutics. 2021, 31 (5), 375-381.
https://doi.org/10.1089/nat.2020.0915

II. Rouven Stulz, Johan Meuller, Dženita Baždarević, Charlotte Wennberg Huldt, Roger Strömberg, Shalini Andersson, Anders Dahlén. A Versatile and Convenient Synthesis of ³⁴S-Labeled Phosphorothioate Oligonucleotides. Chembiochem: a European journal of chemical biology. 2018, 19 (19), 2114-2119.
https://doi.org/10.1002/cbic.201800417

III. 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 Kurczy. NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution. Pharmaceutics. 2022, 14 (2), 463.
https://doi.org/10.3390/pharmaceutics14020463

IV. Kristina Karalė, Martin Bollmark, Rouven Stulz, Dmytro Honcharenko, Ulf Tedebark, Roger Strömberg. A Study on Synthesis and Upscaling of 2′-O-AECM-5-methyl Pyrimidine Phosphoramidites for Oligonucleotide Synthesis. Molecules. 2021, 26 (22), 6927.
https://doi.org/10.3390/molecules26226927

V. Rouven Stulz, Kristina Drucekaite, Alessandro Bonetti, Alexandra Madsen, Shalini Andersson, Anders Dahlén, Roger Strömberg, Synthesis and biological evaluation of 2′-O-(N-Aminoethylcarbamoylmethyl) modified gapmers. [Manuscript]