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Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by gener...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Mary Ann Liebert, Inc., publishers
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997716/ https://www.ncbi.nlm.nih.gov/pubmed/33567244 http://dx.doi.org/10.1089/nat.2020.0907 |
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author | Tone, Yuichiro Mamchaoui, Kamel Tsoumpra, Maria K. Hashimoto, Yasumasa Terada, Reiko Maruyama, Rika Gait, Michael J. Arzumanov, Andrey A. McClorey, Graham Imamura, Michihiro Takeda, Shin'ichi Yokota, Toshifumi Wood, Matthew J.A. Mouly, Vincent Aoki, Yoshitsugu |
author_facet | Tone, Yuichiro Mamchaoui, Kamel Tsoumpra, Maria K. Hashimoto, Yasumasa Terada, Reiko Maruyama, Rika Gait, Michael J. Arzumanov, Andrey A. McClorey, Graham Imamura, Michihiro Takeda, Shin'ichi Yokota, Toshifumi Wood, Matthew J.A. Mouly, Vincent Aoki, Yoshitsugu |
author_sort | Tone, Yuichiro |
collection | PubMed |
description | Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMD(J)) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools. |
format | Online Article Text |
id | pubmed-7997716 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Mary Ann Liebert, Inc., publishers |
record_format | MEDLINE/PubMed |
spelling | pubmed-79977162021-03-29 Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides Tone, Yuichiro Mamchaoui, Kamel Tsoumpra, Maria K. Hashimoto, Yasumasa Terada, Reiko Maruyama, Rika Gait, Michael J. Arzumanov, Andrey A. McClorey, Graham Imamura, Michihiro Takeda, Shin'ichi Yokota, Toshifumi Wood, Matthew J.A. Mouly, Vincent Aoki, Yoshitsugu Nucleic Acid Ther Original Papers Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMD(J)) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools. Mary Ann Liebert, Inc., publishers 2021-04-01 2021-03-25 /pmc/articles/PMC7997716/ /pubmed/33567244 http://dx.doi.org/10.1089/nat.2020.0907 Text en © Yuichiro Tone, et al. 2021; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License [CC-BY] (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Papers Tone, Yuichiro Mamchaoui, Kamel Tsoumpra, Maria K. Hashimoto, Yasumasa Terada, Reiko Maruyama, Rika Gait, Michael J. Arzumanov, Andrey A. McClorey, Graham Imamura, Michihiro Takeda, Shin'ichi Yokota, Toshifumi Wood, Matthew J.A. Mouly, Vincent Aoki, Yoshitsugu Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides |
title | Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides |
title_full | Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides |
title_fullStr | Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides |
title_full_unstemmed | Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides |
title_short | Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides |
title_sort | immortalized canine dystrophic myoblast cell lines for development of peptide-conjugated splice-switching oligonucleotides |
topic | Original Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997716/ https://www.ncbi.nlm.nih.gov/pubmed/33567244 http://dx.doi.org/10.1089/nat.2020.0907 |
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