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Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic...

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Autores principales: Migawa, Michael T, Shen, Wen, Wan, W Brad, Vasquez, Guillermo, Oestergaard, Michael E, Low, Audrey, De Hoyos, Cheryl L, Gupta, Ruchi, Murray, Susan, Tanowitz, Michael, Bell, Melanie, Nichols, Joshua G, Gaus, Hans, Liang, Xue-hai, Swayze, Eric E, Crooke, Stanley T, Seth, Punit P
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582325/
https://www.ncbi.nlm.nih.gov/pubmed/31034558
http://dx.doi.org/10.1093/nar/gkz247
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author Migawa, Michael T
Shen, Wen
Wan, W Brad
Vasquez, Guillermo
Oestergaard, Michael E
Low, Audrey
De Hoyos, Cheryl L
Gupta, Ruchi
Murray, Susan
Tanowitz, Michael
Bell, Melanie
Nichols, Joshua G
Gaus, Hans
Liang, Xue-hai
Swayze, Eric E
Crooke, Stanley T
Seth, Punit P
author_facet Migawa, Michael T
Shen, Wen
Wan, W Brad
Vasquez, Guillermo
Oestergaard, Michael E
Low, Audrey
De Hoyos, Cheryl L
Gupta, Ruchi
Murray, Susan
Tanowitz, Michael
Bell, Melanie
Nichols, Joshua G
Gaus, Hans
Liang, Xue-hai
Swayze, Eric E
Crooke, Stanley T
Seth, Punit P
author_sort Migawa, Michael T
collection PubMed
description Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5′-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.
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spelling pubmed-65823252019-06-21 Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins Migawa, Michael T Shen, Wen Wan, W Brad Vasquez, Guillermo Oestergaard, Michael E Low, Audrey De Hoyos, Cheryl L Gupta, Ruchi Murray, Susan Tanowitz, Michael Bell, Melanie Nichols, Joshua G Gaus, Hans Liang, Xue-hai Swayze, Eric E Crooke, Stanley T Seth, Punit P Nucleic Acids Res NAR Breakthrough Article Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5′-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions. Oxford University Press 2019-06-20 2019-04-29 /pmc/articles/PMC6582325/ /pubmed/31034558 http://dx.doi.org/10.1093/nar/gkz247 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle NAR Breakthrough Article
Migawa, Michael T
Shen, Wen
Wan, W Brad
Vasquez, Guillermo
Oestergaard, Michael E
Low, Audrey
De Hoyos, Cheryl L
Gupta, Ruchi
Murray, Susan
Tanowitz, Michael
Bell, Melanie
Nichols, Joshua G
Gaus, Hans
Liang, Xue-hai
Swayze, Eric E
Crooke, Stanley T
Seth, Punit P
Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins
title Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins
title_full Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins
title_fullStr Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins
title_full_unstemmed Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins
title_short Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins
title_sort site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer asos by modulating interactions with cellular proteins
topic NAR Breakthrough Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582325/
https://www.ncbi.nlm.nih.gov/pubmed/31034558
http://dx.doi.org/10.1093/nar/gkz247
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