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

Vincent Mouly,Shin'Ichi Takeda,Matthew J.A Wood,Yasumasa Hashimoto,Rika Maruyama,Maria K Tsoumpra,Toshifumi Yokota,Michihiro Imamura,Graham Mcclorey,Michael J Gait,Kamel Mamchaoui,Reiko Terada,Yoshitsugu Aoki,Andrey A Arzumanov,Yuichiro Tone

doi:10.1089/nat.2020.0907

Abstract

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.

Highlights

Duchenne muscular dystrophy (DMD) is an X chromosome-linked, progressive, fatal degenerative muscle disorder with an estimated prevalence of 1 in 3,500–5,000 live human male births worldwide [1,2]
There has been a surge in the field of translational research aiming to develop novel gene-based therapeutic approaches for treating DMD, given the fact that current treatment regimens only alleviate the severity or delay the progression of DMD, without successfully reverting the pathophysiological phenotype or eradicating the cause of this disease
Exon skipping has emerged as a promising therapeutic approach for the treatment of patients with DMD; the clinical benefit of the three Food and Drug Administration (FDA)-approved phosphorodiamidate morpholino oligomers (PMOs), eteplirsen, golodirsen, and viltolarsen, requires further confirmatory studies [37]

Summary

Introduction

Duchenne muscular dystrophy (DMD) is an X chromosome-linked, progressive, fatal degenerative muscle disorder with an estimated prevalence of 1 in 3,500–5,000 live human male births worldwide [1,2]. Exon skipping is a novel therapeutic approach that involves the implementation of short, synthetic splice-switching oligonucleotides (SSOs) to effectively target individual DMD gene mutations and restore the DMD reading frame [5,6]. VILTEPSOÒ (viltolarsen), an antisense oligonucleotide drug codeveloped by the National Center of Neurology and Psychiatry (NCNP) in collaboration with Nippon Shinyaku Co., Ltd., which targets DMD patients with confirmed mutations amenable to exon 53 skipping, has received conditional approval in both Japan and the United States, and is awaiting the results of phase 3 trial (NCT04060199) to confirm its efficacy and clinical benefit [9,10,11]

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