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

Nature Biotechnology volume 37, pages 640–650 (2019)Cite this article

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Abstract

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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Fig. 1: Toxic cEt gapmer PS-ASOs induce apoptosis.
Fig. 2: Toxic 3-10-3 cEt gapmer PS-ASOs delocalize paraspeckle proteins and kinetics of ASO toxicity.
Fig. 3: Toxicity of PS-ASOs correlates with intracellular protein-binding.
Fig. 4: A single 2′-OMe modification at gap position 2 reduces ASO–protein interactions and mitigates ASO toxicity.
Fig. 5: The 2′-OMe modification at gap position 2 broadly mitigates toxicity and improves the therapeutic index of PS-ASOs.
Fig. 6: Models of PS-ASO toxicity.

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Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files.

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Acknowledgements

This work was supported by Ionis Pharmaceuticals internal funding. We thank B. DeBrosse-Serra and X. Xiao for histology assistance; M. Andrade and T. Prakash for ASO synthesis; N. Allen for help in the animal studies; D. Sipe for handling of animal facilities; A. Berdeja for technical support; T. Reigle for helping with figure preparation; and H. Chang, S. Wang, J. Bailey, M. Graham, A. Mullick, E. Swayze and F. Bennett for discussions.

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Authors and Affiliations

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

    Wen Shen, Cheryl L. De Hoyos, Michael T. Migawa, Timothy A. Vickers, Hong Sun, Audrey Low, Thomas A. Bell III, Meghdad Rahdar, Swagatam Mukhopadhyay, Christopher E. Hart, Melanie Bell, Stan Riney, Susan F. Murray, Sarah Greenlee, Rosanne M. Crooke, Xue-hai Liang, Punit P. Seth & Stanley T. Crooke

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Contributions

W.S., M.T.M., P.P.S., T.A.V., X.L. and S.T.C. designed the research. W.S., C.L.D., M.T.M., T.A.V., H.S., A.L., T.A.B.III, M.R., S.M., C.E.H., M.B., S.F.M., R.M.C. and S.G. performed the experiments. All authors analyzed the data. W.S., P.P.S., T.A.V., X.L. and S.T.C. wrote the manuscript.

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Correspondence to Stanley T. Crooke.

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All authors are employees of Ionis Pharmaceuticals. A patent related to this study has been submitted.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–42 and Supplementary Tables 1–7

Reporting Summary

Supplementary Video 1

Kinetics of non-toxic cEt gapmer PS-ASOs association with P54nrb in live cells. The HeLa/LgBiT-P54nrb cell line was transfected with pNPM1-GFP. The following day cells were treated with 100 nM non-toxic SmBiT-conjugated cEt gapmer PS-ASO 978671 (3′ SmBiT peptide-conjugated 549148) and visualized using a bioluminescence imaging microscope at 3-min intervals for a total of 2.25 h collecting brightfield, GFP (green) and NLuc bioluminescence (blue) signals. Data are representative of three biologically independent experiments.

Supplementary Video 2

Kinetics of toxic cEt gapmer PS-ASOs association with P54nrb in live cells. The HeLa/LgBiT-P54nrb cell line was transfected with pNPM1-GFP. The following day cells were treated with 100 nM toxic SmBiT-conjugated cEt gapmer PS-ASO 978780 (3′ SmBiT peptide-conjugated 464917) and visualized using a bioluminescence imaging microscope at 3-min intervals for a total of 1.5 h collecting brightfield, GFP (green) and NLuc bioluminescence (blue) signals. Data are representative of three biologically independent experiments.

Supplementary Video 3

Live cell movie of toxic PS-ASO-induced nucleolar delocalization of paraspeckle protein PSF. HeLa cells overexpressing GFP-PSF (white signal) were transfected with toxic (T) PS-ASO 821033 (5′-Cy3-labeled 558807; red signal) at 200 nM and visualized using a confocal microscope at 5-min intervals for 70 minutes. Data are representative of three biologically independent experiments.

Supplementary Video 4

Kinetics of toxic cEt gapmer PS-ASO association with P54nrb in live cells depleted of RNase H1. The LgBiT-P54nrb HeLa cell line was treated with a siRNA targeting RNase H1 for 48 h. The cells were then treated with the toxic SmBiT-conjugated cEt ASO 978780 at 100 nM. Images were collected at 3-minute intervals for 2 h collecting brightfield and NLuc bioluminescence (blue) signals. Data are representative of three biologically independent experiments.

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Shen, W., De Hoyos, C.L., Migawa, M.T. et al. Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index. Nat Biotechnol 37, 640–650 (2019). https://doi.org/10.1038/s41587-019-0106-2

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