4.3 Article

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides

Journal

NUCLEIC ACID THERAPEUTICS
Volume 31, Issue 2, Pages 172-181

Publisher

MARY ANN LIEBERT, INC
DOI: 10.1089/nat.2020.0907

Keywords

Duchenne muscular dystrophy; canine X-linked muscular dystrophy in Japan (CXMDJ); immortalized dystrophic canine myoblast; splice-switching oligonucleotides; phosphorodiamidate morpholino oligomer; cell-penetrating peptide

Funding

  1. Japan Society for the Promotion of Science [18K07544, 28-6]
  2. Japan Agency for Medical Research and Development [18ek0109239h0002, 18lm0203066h0001, 18lm0203069h0001]
  3. Association Francaise Contre les Myopathies (AFM)
  4. Nippon Shinyaku Co., Ltd.
  5. MRC [MC_U105178803] Funding Source: UKRI

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Researchers have established immortalized canine myoblast lines to demonstrate that a novel cell-penetrating peptide-conjugated splice-switching oligonucleotide significantly improves multiexon skipping activity. This approach could serve as a basis for future pharmacological studies on drug delivery tools.
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.

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