Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle

Abstract Exon skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), employing morpholino antisense oligonucleotides (PMO-AO) to exclude disruptive exons from the mutant DMD transcript and elicit production of truncated dystrophin protein. Clinical trials for PMO show variable and sporadic dystrophin rescue. Here, we show that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. PMO localization is sustained in inflammatory foci where it enters macrophages, actively differentiating myoblasts and newly forming myotubes. We conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers. Identification of these factors accounts for the variability in clinical trials and suggests strategies to improve this therapeutic approach to DMD.

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New Approach for Antisense Oligonucleotide-Mediated Exon Skipping in Duchenne Muscular Dystrophy

Journal of Advanced Computational Intelligence and Intelligent Informatics ◽

10.20965/jaciii.2012.p0521 ◽

2012 ◽

Vol 16 (4) ◽

pp. 521-526

Author(s):

Yoshitsugu Aoki ◽

◽

Tetsuya Nagata ◽

Shin’ichi Takeda

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Exon Skipping ◽

New Approach ◽

Reading Frame ◽

Promising Therapeutic Approach ◽

Dmd Gene ◽

Dystrophin Protein

Duchenne Muscular Dystrophy (DMD) is a lethalmuscle disorder characterized by mutations in the DMD gene. These mutations primarily disrupt the reading frame, resulting in the absence of functional dystrophin protein. Exon skipping, which involves the use of antisense oligonucleotides is a promising therapeutic approach for DMD, and clinical trials on exon skipping are currently underway in DMD patients. Recently, stable and less-toxic antisense oligonucleotides with higher efficacy have been developed in mouse and dog models of DMD. This review highlights a new approach for antisense oligonucleotide-based therapeutics for DMD, particularly for exon skipping-based methods.

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The administration of antisense oligonucleotide golodirsen reduces pathological regeneration in patients with Duchenne muscular dystrophy

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01106-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Dominic Scaglioni ◽

Francesco Catapano ◽

Matthew Ellis ◽

Silvia Torelli ◽

Darren Chambers ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Antisense Oligonucleotide ◽

De Novo ◽

Exon Skipping ◽

Significant Negative Correlation ◽

Entire Cohort ◽

Associated Proteins ◽

Analysis Platform ◽

Dystrophin Protein

AbstractDuring the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.

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In vivocomparison of 2′-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping

The Journal of Gene Medicine ◽

10.1002/jgm.1288 ◽

2009 ◽

Vol 11 (3) ◽

pp. 257-266 ◽

Cited By ~ 128

Author(s):

Hans A. Heemskerk ◽

Christa L. de Winter ◽

Sjef J. de Kimpe ◽

Petra van Kuik-Romeijn ◽

Niki Heuvelmans ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Antisense Oligonucleotides ◽

Exon Skipping

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The Interplay of Mitophagy and Inflammation in Duchenne Muscular Dystrophy

Life ◽

10.3390/life11070648 ◽

2021 ◽

Vol 11 (7) ◽

pp. 648

Author(s):

Andrea L. Reid ◽

Matthew S. Alexander

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mitochondrial Dysfunction ◽

Disease Onset ◽

Exon Skipping ◽

Dystrophin Gene ◽

Muscle Disease ◽

Mitochondrial Morphology ◽

Gene Therapies ◽

Dystrophin Protein

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a pathogenic disruption of the DYSTROPHIN gene that results in non-functional dystrophin protein. DMD patients experience loss of ambulation, cardiac arrhythmia, metabolic syndrome, and respiratory failure. At the molecular level, the lack of dystrophin in the muscle results in myofiber death, fibrotic infiltration, and mitochondrial dysfunction. There is no cure for DMD, although dystrophin-replacement gene therapies and exon-skipping approaches are being pursued in clinical trials. Mitochondrial dysfunction is one of the first cellular changes seen in DMD myofibers, occurring prior to muscle disease onset and progresses with disease severity. This is seen by reduced mitochondrial function, abnormal mitochondrial morphology and impaired mitophagy (degradation of damaged mitochondria). Dysfunctional mitochondria release high levels of reactive oxygen species (ROS), which can activate pro-inflammatory pathways such as IL-1β and IL-6. Impaired mitophagy in DMD results in increased inflammation and further aggravates disease pathology, evidenced by increased muscle damage and increased fibrosis. This review will focus on the critical interplay between mitophagy and inflammation in Duchenne muscular dystrophy as a pathological mechanism, as well as describe both candidate and established therapeutic targets that regulate these pathways.

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Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy

Neurology ◽

10.1212/wnl.0000000000009233 ◽

2020 ◽

Vol 94 (21) ◽

pp. e2270-e2282 ◽

Cited By ~ 25

Author(s):

Diane E. Frank ◽

Frederick J. Schnell ◽

Cody Akana ◽

Saleh H. El-Husayni ◽

Cody A. Desjardins ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Western Blot ◽

Exon Skipping ◽

Study Drug ◽

Dose Titration ◽

Open Label ◽

Double Blind ◽

Dystrophin Protein ◽

Muscle Biopsies

ObjectiveTo report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping.MethodsPart 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry.ResultsTwelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%–4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry.ConclusionGolodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization.Clinicaltrials.gov identifierNCT02310906.Classification of evidenceThis study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

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