Antisense PMO cocktails effectively skip dystrophin exons 45-55 in myotubes transdifferentiated from DMD patient fibroblasts.

William Duddy,Toshifumi Yokota,Yusuke Echigoya,Takashi Saito,Yoshitsugu Aoki,Joshua Lee,Shin’Ichi Takeda,Atsushi Asakura

doi:10.1371/journal.pone.0197084

Abstract

Antisense-mediated exon skipping has made significant progress as a therapeutic platform in recent years, especially in the case of Duchenne muscular dystrophy (DMD). Despite FDA approval of eteplirsen–the first-ever antisense drug clinically marketed for DMD–exon skipping therapy still faces the significant hurdles of limited applicability and unknown truncated protein function. In-frame exon skipping of dystrophin exons 45–55 represents a significant approach to treating DMD, as a large proportion of patients harbor mutations within this “hotspot” region. Additionally, patients harboring dystrophin exons 45–55 deletion mutations are reported to have exceptionally mild to asymptomatic phenotypes. Here, we demonstrate that a cocktail of phosphorodiamidate morpholino oligomers can effectively skip dystrophin exons 45–55 in vitro in myotubes transdifferentiated from DMD patient fibroblast cells. This is the first report of substantive exons 45–55 skipping in DMD patient cells. These findings help validate the use of transdifferentiated patient fibroblast cells as a suitable cell model for dystrophin exon skipping assays and further emphasize the feasibility of dystrophin exons 45–55 skipping in patients.

Highlights

Duchenne muscular dystrophy (DMD) is a lethal, progressive myopathy affecting approximately 1 in every 3600–5000 male births and is caused by deleterious mutations in the dystrophin (DMD) gene [1,2,3,4]
We demonstrated the feasibility of skipping dystrophin exons 45–55 in vitro using human DMD patients’ myotubes converted from fibroblasts
This is the first successful demonstration of robust, dose-dependent dystrophin exons 45–55 skipping in DMD patient cells

Summary

Introduction

Duchenne muscular dystrophy (DMD) is a lethal, progressive myopathy affecting approximately 1 in every 3600–5000 male births and is caused by deleterious mutations in the dystrophin (DMD) gene [1,2,3,4]. Mutations in DMD can cause another milder form of muscular dystrophy known as Becker muscular dystrophy (BMD) [5]. DMD arises from outof-frame mutations (~65% of patients) while BMD generally arises from in-frame mutations (~82% of patients) [6,7,8]. Therapy, the Women and Children’s Health Research Institute (WCHRI), and Alberta Innovates Health Solutions (AIHS)

Methods

Results

Conclusion