Exons 45–55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene

Abstract

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO-cocktail sets for exons 45-55 skipping aiming to produce a dystrophin form with preserved functionality as seen in milder/asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs of which each efficiently skips an assigned exon in cell-based screening. Its combinational PMOs fitted to different deletions of immortalized DMD patient-muscle cells significantly induced exons 45-55-skipped transcripts with removing three, eight or ten exons and dystrophin restoration as represented by Western blotting. In vivo skipping of the maximum eleven human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used as mutation-tailored cocktails for exons 45-55 skipping and treat over 65% DMD patients carrying out-of-or in-frame deletions.

A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy

“…Restoring the ORF by excluding DMD exons can be executed through antisense oligonucleotide (ASO) treatment or gene editing ( Aartsma-Rus et al, 2009 ; Wong and Cohn, 2017 ). To date, skipping of DMD exons 45-55 using ASO treatment has been studied in patient cells, mdx52 mice (which carry a Dmd exon 52 deletion) and a humanized DMD mouse model to understand the skipping efficiency and/or level of dystrophin restoration ( Aoki et al, 2012 ; Echigoya et al, 2015 , 2019 ; Lee et al, 2018 ; van Vliet et al, 2008 ). However, improvement in cardiac function was not assessed in these studies, because cardiac pathology has not been described in these mouse models.…”

Current Genetic Survey and Potential Gene-Targeting Therapeutics for Neuromuscular Diseases

“…Analyzing out-of-frame deletion mutations, researchers identified exons 45–55 as the dystrophin gene mutation hotspot, accounting for almost 50% of DMD patients [ 21 , 22 , 23 ]. Several AO cocktails achieved skipping of up to 10 exons in vitro and in vivo [ 24 ]. The tailored cocktail combinations offer higher hopes in expanding patients’ coverage with out-of-frame deletions than single exon skipping and enhancing motor dysfunction alleviation.…”

A Genotype-Phenotype Correlation Study of Exon Skip-Equivalent In-Frame Deletions and Exon Skip-Amenable Out-of-Frame Deletions across the DMD Gene to Simulate the Effects of Exon-Skipping Therapies: A Meta-Analysis

“…However, because of these drugs’ highly specific nature, they apply to only a small portion of the DMD population, e.g., eteplirsen, golodirsen, and viltolarsen together are applicable for a total of around 20% of the entire DMD population in the U.S. [ 12 , 13 ]. Recently, skipping of multiple exons, e.g., exons 45–55, using mutation-tailored cocktails of antisense oligonucleotides have shown potential for clinical application, and the applicability of such cocktail drug was shown to reach over 65% of DMD patients with large deletions [ 6 , 15 ].…”