Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.
Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.
Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry.
Bailey
B
Miskew Nichols
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry.
Yoshitsugu
Y
Aoki
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry.
Mutsuki
M
Kuraoka
Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry.
Joshua J A
JJ
Lee
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry; takeda@ncnp.go.jp.
Shin'ichi
S
Takeda
Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry; toshifum@ualberta.ca.
Toshifumi
T
Yokota
D000818:Animals; D004195:Disease Models, Animal; D004285:Dogs; D016189:Dystrophin / Q000235:genetics; D005091:Exons; D015316:Genetic Therapy / Q000379:methods; D060172:Morpholinos / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D009137:Muscular Dystrophy, Animal / Q000235:genetics* / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D016189:Dystrophin; D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger