4.3 Article

Evaluation of Phosphorus and Non-Phosphorus Neutral Oligonucleotide Backbones for Enhancing Therapeutic Index of Gapmer Antisense Oligonucleotides

Journal

NUCLEIC ACID THERAPEUTICS
Volume 32, Issue 1, Pages 40-50

Publisher

MARY ANN LIEBERT, INC
DOI: 10.1089/nat.2021.0064

Keywords

therapeutic index; gapmer ASO; neutral backbone; activity

Funding

  1. Ionis Pharmaceuticals, Inc.

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The phosphorothioate (PS) linkage plays a crucial role in therapeutic oligonucleotides by enhancing therapeutic index. The exploration of phosphorus- and non-phosphorus-based neutral backbone modifications can improve the activity of neutral linkages, but factors such as chemical stability and properties need to be considered.
The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

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