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Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of resto...

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Autores principales: Novak, James S., Spathis, Rita, Dang, Utkarsh J., Fiorillo, Alyson A., Hindupur, Ravi, Tully, Christopher B., Mázala, Davi A.G., Canessa, Emily, Brown, Kristy J., Partridge, Terence A., Hathout, Yetrib, Nagaraju, Kanneboyina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: IOS Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8673539/
https://www.ncbi.nlm.nih.gov/pubmed/34569969
http://dx.doi.org/10.3233/JND-210696
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author Novak, James S.
Spathis, Rita
Dang, Utkarsh J.
Fiorillo, Alyson A.
Hindupur, Ravi
Tully, Christopher B.
Mázala, Davi A.G.
Canessa, Emily
Brown, Kristy J.
Partridge, Terence A.
Hathout, Yetrib
Nagaraju, Kanneboyina
author_facet Novak, James S.
Spathis, Rita
Dang, Utkarsh J.
Fiorillo, Alyson A.
Hindupur, Ravi
Tully, Christopher B.
Mázala, Davi A.G.
Canessa, Emily
Brown, Kristy J.
Partridge, Terence A.
Hathout, Yetrib
Nagaraju, Kanneboyina
author_sort Novak, James S.
collection PubMed
description Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.
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spelling pubmed-86735392021-12-29 Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy Novak, James S. Spathis, Rita Dang, Utkarsh J. Fiorillo, Alyson A. Hindupur, Ravi Tully, Christopher B. Mázala, Davi A.G. Canessa, Emily Brown, Kristy J. Partridge, Terence A. Hathout, Yetrib Nagaraju, Kanneboyina J Neuromuscul Dis Research Article Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD. IOS Press 2021-11-30 /pmc/articles/PMC8673539/ /pubmed/34569969 http://dx.doi.org/10.3233/JND-210696 Text en © 2021 – The authors. Published by IOS Press https://creativecommons.org/licenses/by-nc/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial (CC BY-NC 4.0) License (https://creativecommons.org/licenses/by-nc/4.0/) , which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Novak, James S.
Spathis, Rita
Dang, Utkarsh J.
Fiorillo, Alyson A.
Hindupur, Ravi
Tully, Christopher B.
Mázala, Davi A.G.
Canessa, Emily
Brown, Kristy J.
Partridge, Terence A.
Hathout, Yetrib
Nagaraju, Kanneboyina
Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy
title Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy
title_full Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy
title_fullStr Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy
title_full_unstemmed Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy
title_short Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy
title_sort interrogation of dystrophin and dystroglycan complex protein turnover after exon skipping therapy
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8673539/
https://www.ncbi.nlm.nih.gov/pubmed/34569969
http://dx.doi.org/10.3233/JND-210696
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