Open AccessImmortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides
Author(s) -
Yukiko Tone,
Kamel Mamchaoui,
M.K. Tsoumpra,
Yasumasa Hashimoto,
Reiko Terada,
Rika Maruyama,
Michael J. Gait,
Andrey A. Arzumanov,
Graham McClorey,
Michihiro Imamura,
Shin’ichi Takeda,
Toshifumi Yokota,
Matthew J. Wood,
Vincent Mouly,
Yosuke Aoki
Publication year - 2021
Publication title -
nucleic acid therapeutics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.255
H-Index - 67
eISSN - 2159-3345
pISSN - 2159-3337
DOI - 10.1089/nat.2020.0907
Subject(s) - duchenne muscular dystrophy , morpholino , dystrophin , myogenesis , exon skipping , biology , myocyte , microbiology and biotechnology , immortalised cell line , cell culture , exon , genetics , alternative splicing , gene knockdown , gene
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.