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AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1

Expansion of an unstable CTG repeat in the 3′UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUG(exp)) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splici...

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Autores principales: Stepniak-Konieczna, Ewa, Konieczny, Patryk, Cywoniuk, Piotr, Dluzewska, Julia, Sobczak, Krzysztof
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049696/
https://www.ncbi.nlm.nih.gov/pubmed/31965181
http://dx.doi.org/10.1093/nar/gkaa007
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author Stepniak-Konieczna, Ewa
Konieczny, Patryk
Cywoniuk, Piotr
Dluzewska, Julia
Sobczak, Krzysztof
author_facet Stepniak-Konieczna, Ewa
Konieczny, Patryk
Cywoniuk, Piotr
Dluzewska, Julia
Sobczak, Krzysztof
author_sort Stepniak-Konieczna, Ewa
collection PubMed
description Expansion of an unstable CTG repeat in the 3′UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUG(exp)) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUG(exp), we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUG(exp) from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUG(exp). Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs’ chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1.
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spelling pubmed-70496962020-03-10 AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1 Stepniak-Konieczna, Ewa Konieczny, Patryk Cywoniuk, Piotr Dluzewska, Julia Sobczak, Krzysztof Nucleic Acids Res Molecular Biology Expansion of an unstable CTG repeat in the 3′UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUG(exp)) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUG(exp), we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUG(exp) from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUG(exp). Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs’ chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1. Oxford University Press 2020-03-18 2020-01-22 /pmc/articles/PMC7049696/ /pubmed/31965181 http://dx.doi.org/10.1093/nar/gkaa007 Text en © The Author(s) 2020. 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
Stepniak-Konieczna, Ewa
Konieczny, Patryk
Cywoniuk, Piotr
Dluzewska, Julia
Sobczak, Krzysztof
AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1
title AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1
title_full AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1
title_fullStr AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1
title_full_unstemmed AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1
title_short AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1
title_sort aon-induced splice-switching and dmpk pre-mrna degradation as potential therapeutic approaches for myotonic dystrophy type 1
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049696/
https://www.ncbi.nlm.nih.gov/pubmed/31965181
http://dx.doi.org/10.1093/nar/gkaa007
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