Journal article
Fine tuning of phosphorothioate inclusion in 2'-O-methyl oligonucleotides contributes to specific cell targeting for splice-switching modulation
- Abstract:
- 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-<i>mdx</i> 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 <i>via</i> 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, <i>Ncl</i> 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.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
Actions
Access Document
- Files:
-
-
(Preview, Version of record, pdf, 1.4MB, Terms of use)
-
- Publisher copy:
- 10.3389/fphys.2021.689179
Authors
- Publisher:
- Frontiers Media
- Journal:
- Frontiers in Physiology More from this journal
- Volume:
- 12
- Article number:
- 689179
- Place of publication:
- Switzerland
- Publication date:
- 2021-10-13
- Acceptance date:
- 2021-09-06
- DOI:
- EISSN:
-
1664-042X
- ISSN:
-
1664-042X
- Pmid:
-
34721051
- Language:
-
English
- Keywords:
- Pubs id:
-
1207792
- Local pid:
-
pubs:1207792
- Deposit date:
-
2023-09-21
Terms of use
- Copyright holder:
- Aoki et al
- Copyright date:
- 2021
- Rights statement:
- © 2021 Aoki, Rocha, Lehto, Miyatake, Johansson, Hashimoto, Nordin, Mager, Aoki, Graham, Sathyaprakash, Roberts, Wood, Behlke and Andaloussi. 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.
- Licence:
- CC Attribution (CC BY)
If you are the owner of this record, you can report an update to it here: Report update to this record