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 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 (CXMDJ) 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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:31 |
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| Enthalten in: |
Nucleic acid therapeutics - 31(2021), 2 vom: 01. Apr., Seite 172-181 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Tone, Yuichiro [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 22.11.2021 Date Revised 15.11.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1089/nat.2020.0907 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM321273567 |
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| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 (CXMDJ) 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Duchenne muscular dystrophy | |
| 650 | 4 | |a canine X-linked muscular dystrophy in Japan (CXMDJ) | |
| 650 | 4 | |a cell-penetrating peptide | |
| 650 | 4 | |a immortalized dystrophic canine myoblast | |
| 650 | 4 | |a phosphorodiamidate morpholino oligomer | |
| 650 | 4 | |a splice-switching oligonucleotides | |
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| 700 | 1 | |a Mamchaoui, Kamel |e verfasserin |4 aut | |
| 700 | 1 | |a Tsoumpra, Maria K |e verfasserin |4 aut | |
| 700 | 1 | |a Hashimoto, Yasumasa |e verfasserin |4 aut | |
| 700 | 1 | |a Terada, Reiko |e verfasserin |4 aut | |
| 700 | 1 | |a Maruyama, Rika |e verfasserin |4 aut | |
| 700 | 1 | |a Gait, Michael J |e verfasserin |4 aut | |
| 700 | 1 | |a Arzumanov, Andrey A |e verfasserin |4 aut | |
| 700 | 1 | |a McClorey, Graham |e verfasserin |4 aut | |
| 700 | 1 | |a Imamura, Michihiro |e verfasserin |4 aut | |
| 700 | 1 | |a Takeda, Shin'ichi |e verfasserin |4 aut | |
| 700 | 1 | |a Yokota, Toshifumi |e verfasserin |4 aut | |
| 700 | 1 | |a Wood, Matthew J A |e verfasserin |4 aut | |
| 700 | 1 | |a Mouly, Vincent |e verfasserin |4 aut | |
| 700 | 1 | |a Aoki, Yoshitsugu |e verfasserin |4 aut | |
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