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Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation
Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistr...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8548633/ https://www.ncbi.nlm.nih.gov/pubmed/34721051 http://dx.doi.org/10.3389/fphys.2021.689179 |
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author | Aoki, Yoshitsugu Rocha, Cristina S. J. Lehto, Taavi Miyatake, Shouta Johansson, Henrik Hashimoto, Yasumasa Nordin, Joel Z. Mager, Imre Aoki, Misako Graham, McClorey Sathyaprakash, Chaitra Roberts, Thomas C. Wood, Matthew J. A. Behlke, Mark A. Andaloussi, Samir El |
author_facet | Aoki, Yoshitsugu Rocha, Cristina S. J. Lehto, Taavi Miyatake, Shouta Johansson, Henrik Hashimoto, Yasumasa Nordin, Joel Z. Mager, Imre Aoki, Misako Graham, McClorey Sathyaprakash, Chaitra Roberts, Thomas C. Wood, Matthew J. A. Behlke, Mark A. Andaloussi, Samir El |
author_sort | Aoki, Yoshitsugu |
collection | PubMed |
description | Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery. |
format | Online Article Text |
id | pubmed-8548633 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-85486332021-10-28 Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation Aoki, Yoshitsugu Rocha, Cristina S. J. Lehto, Taavi Miyatake, Shouta Johansson, Henrik Hashimoto, Yasumasa Nordin, Joel Z. Mager, Imre Aoki, Misako Graham, McClorey Sathyaprakash, Chaitra Roberts, Thomas C. Wood, Matthew J. A. Behlke, Mark A. Andaloussi, Samir El Front Physiol Physiology Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery. Frontiers Media S.A. 2021-10-13 /pmc/articles/PMC8548633/ /pubmed/34721051 http://dx.doi.org/10.3389/fphys.2021.689179 Text en Copyright © 2021 Aoki, Rocha, Lehto, Miyatake, Johansson, Hashimoto, Nordin, Mager, Aoki, Graham, Sathyaprakash, Roberts, Wood, Behlke and Andaloussi. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Physiology Aoki, Yoshitsugu Rocha, Cristina S. J. Lehto, Taavi Miyatake, Shouta Johansson, Henrik Hashimoto, Yasumasa Nordin, Joel Z. Mager, Imre Aoki, Misako Graham, McClorey Sathyaprakash, Chaitra Roberts, Thomas C. Wood, Matthew J. A. Behlke, Mark A. Andaloussi, Samir El Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation |
title | Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation |
title_full | Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation |
title_fullStr | Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation |
title_full_unstemmed | Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation |
title_short | Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation |
title_sort | fine tuning of phosphorothioate inclusion in 2′-o-methyl oligonucleotides contributes to specific cell targeting for splice-switching modulation |
topic | Physiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8548633/ https://www.ncbi.nlm.nih.gov/pubmed/34721051 http://dx.doi.org/10.3389/fphys.2021.689179 |
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