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Nat Biotechnol. 2019 Jun;37(6):640-650. doi: 10.1038/s41587-019-0106-2. Epub 2019 Apr 29.

Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.

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Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA.
Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA.


The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2'-O-methoxyethyl (2'-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1-dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2'-O-methyl (2'-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles.

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