Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
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
_version_ 1784590616471011328
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
work_keys_str_mv AT aokiyoshitsugu finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT rochacristinasj finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT lehtotaavi finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT miyatakeshouta finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT johanssonhenrik finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT hashimotoyasumasa finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT nordinjoelz finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT magerimre finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT aokimisako finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT grahammcclorey finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT sathyaprakashchaitra finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT robertsthomasc finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT woodmatthewja finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT behlkemarka finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation
AT andaloussisamirel finetuningofphosphorothioateinclusionin2omethyloligonucleotidescontributestospecificcelltargetingforspliceswitchingmodulation