Cargando…
Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides
Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15–25 nt l...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954394/ https://www.ncbi.nlm.nih.gov/pubmed/31802121 http://dx.doi.org/10.1093/nar/gkz1132 |
_version_ | 1783486788666392576 |
---|---|
author | Scharner, Juergen Ma, Wai Kit Zhang, Qian Lin, Kuan-Ting Rigo, Frank Bennett, C Frank Krainer, Adrian R |
author_facet | Scharner, Juergen Ma, Wai Kit Zhang, Qian Lin, Kuan-Ting Rigo, Frank Bennett, C Frank Krainer, Adrian R |
author_sort | Scharner, Juergen |
collection | PubMed |
description | Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15–25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events. |
format | Online Article Text |
id | pubmed-6954394 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-69543942020-01-16 Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides Scharner, Juergen Ma, Wai Kit Zhang, Qian Lin, Kuan-Ting Rigo, Frank Bennett, C Frank Krainer, Adrian R Nucleic Acids Res Molecular Biology Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15–25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events. Oxford University Press 2020-01-24 2019-12-05 /pmc/articles/PMC6954394/ /pubmed/31802121 http://dx.doi.org/10.1093/nar/gkz1132 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 | Molecular Biology Scharner, Juergen Ma, Wai Kit Zhang, Qian Lin, Kuan-Ting Rigo, Frank Bennett, C Frank Krainer, Adrian R Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides |
title | Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides |
title_full | Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides |
title_fullStr | Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides |
title_full_unstemmed | Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides |
title_short | Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides |
title_sort | hybridization-mediated off-target effects of splice-switching antisense oligonucleotides |
topic | Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954394/ https://www.ncbi.nlm.nih.gov/pubmed/31802121 http://dx.doi.org/10.1093/nar/gkz1132 |
work_keys_str_mv | AT scharnerjuergen hybridizationmediatedofftargeteffectsofspliceswitchingantisenseoligonucleotides AT mawaikit hybridizationmediatedofftargeteffectsofspliceswitchingantisenseoligonucleotides AT zhangqian hybridizationmediatedofftargeteffectsofspliceswitchingantisenseoligonucleotides AT linkuanting hybridizationmediatedofftargeteffectsofspliceswitchingantisenseoligonucleotides AT rigofrank hybridizationmediatedofftargeteffectsofspliceswitchingantisenseoligonucleotides AT bennettcfrank hybridizationmediatedofftargeteffectsofspliceswitchingantisenseoligonucleotides AT kraineradrianr hybridizationmediatedofftargeteffectsofspliceswitchingantisenseoligonucleotides |