Morpholino Oligomer–Mediated Exon Skipping Averts the Onset of Dystrophic Pathology in the mdx Mouse

Fletcher, Sue; Honeyman, K.; Fall, Abbie; Harding, Penny; Johnsen, R.; Steinhaus, Joshua P; Moulton, Hong M.; Iversen, Patrick L.; Wilton, Steve D.

doi:10.1038/sj.mt.6300245

Cited by 139 publications

(120 citation statements)

References 41 publications

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“…Effort has been made to improve antisense effect by modification of AONs with cell-penetrating peptides, but with limited success (30, 31). Fletcher et al (31) used a PMO tagged with an arginine-rich peptide for exon 23 skipping in mdx mice and reported restoration of near-normal levels of dystrophin in diaphragm by i.p. injections.…”

Section: Discussionmentioning

confidence: 99%

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Moulton

Iversen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

221

258

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Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cellpenetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.M utations in the dystrophin gene underlie two forms of muscular dystrophy: Duchenne and Becker muscular dystrophy (DMD and BMD). DMD is caused mainly by nonsense and frame-shift mutations with little or no production of functional dystrophin protein, leading to disease onset in early childhood with lethal consequences. BMD is caused by mutations that typically create shortened but in-frame transcripts with production of partially functional dystrophin, leading to variable and often overt symptoms (1-3). Most DMD mutations occur within the rod domain, which spans more than half the length of the protein, but seems to have limited functional importance (4, 5). Antisense therapy uses specific oligomers to remove the mutated or additional exon(s) that disrupt the reading frame, thus restoring the expression of shortened forms of dystrophin protein retaining critical functions (6-11).We previously demonstrated that i.m. delivery of a specific 2Ј-O-methyl phosphorothioate antisense oligonucleotide (2ЈOMeAON) was able to skip targeted dystrophin exon 23 in mdx mouse, a model of DMD (9). This mouse carries a nonsense point mutation within exon 23 and lacks dystrophin expression (except in a few rare revertant fibers) in all muscles, including the heart (12, 13). Skipping the mutated exon 23 restored both the reading frame and dystrophin expression, with functional improvement of the treated muscles (14) [supporting information (SI) Fig. S1a]. Recently we showed that a phosphorodiamidate morpholino oligomer (PMO), E23ϩ7-18 targeting the junction of exon 23 and intron 23 of mouse dystrophin (referred to as PMOE23 hereafter), was able to induce up to functional levels of dystrophin expression in some skeletal muscles by regular i.v. injections in mdx mice (15). However, dystrophin expression induced by both 2ЈOMeAON and PMO required high doses and was highly variable between muscles and myofibers in terms of observed efficacy. Of greater conc...

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Section: Discussionmentioning

confidence: 99%

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Moulton

Iversen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

221

258

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“…This is important given that cardiac complications are observed in up to 90% of DMD patients (102) and that 1/3 of DMD patients suffer cognitive impairment related to the deficiency of dystrophin in the brain (103,104). One approach to improve AO targeting to cardiac muscle is the direct conjugation of cell penetrating peptides to AOs which improves AO delivery to skeletal (105)(106)(107)(108)(109)(110)(111)(112) and cardiac mdx mouse muscles (111,(113)(114)(115); however the toxicology of these conjugates has yet to be ascertained.…”

Section: Next Generation Aosmentioning

confidence: 99%

Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges

Arechavala‐Gomeza¹,

Anthony²,

Morgan³

et al. 2012

CGT

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Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF and the consequent production of a shorter but functional dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD and progress towards clinical application has been rapid. Less than a decade has passed between the first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to cardiac muscle remain significant challenges. Here we review the current status of AO-mediated therapy for DMD, discussing the pre-clinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.

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“…Currently there are no effective therapies for the cardiomyopathy of DMD. Numerous therapeutic strategies have been proposed to impact striated muscle disease progression in DMD (6)(7)(8)(9)(10)(11)(12). Most of these approaches focus primarily on the skeletal muscle manifestations of the disease, often leaving the deteriorating heart untreated (6, 7, 9-11).…”

Section: Introductionmentioning

confidence: 99%

Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs

Townsend¹,

Turner²,

Yasuda³

et al. 2010

J. Clin. Invest.

102

111

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Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration caused by lack of the cytoskeletal protein dystrophin. Dystrophin deficiency causes muscle membrane instability, skeletal muscle wasting, cardiomyopathy, and heart failure. Advances in palliative respiratory care have increased the incidence of heart disease in DMD patients, for which there is no cure or effective therapy. Here we have shown that chronic infusion of membrane-sealing poloxamer to severely affected dystrophic dogs reduced myocardial fibrosis, blocked increased serum cardiac troponin I (cTnI) and brain type natriuretic peptide (BNP), and fully prevented left-ventricular remodeling. Mechanistically, we observed a markedly greater primary defect of reduced cell compliance in dystrophic canine myocytes than in the mildly affected mdx mouse myocytes, and this was associated with a lack of utrophin upregulation in the dystrophic canine cardiac myocytes. Interestingly, after chronic poloxamer treatment, the poor compliance of isolated canine myocytes remained evident, but this could be restored to normal upon direct application of poloxamer. Collectively, these findings indicate that dystrophin and utrophin are critical to membrane stability-dependent cardiac myocyte mechanical compliance and that poloxamer confers a highly effective membrane-stabilizing chemical surrogate in dystrophin/utrophin deficiency. We propose that membrane sealant therapy is a potential treatment modality for DMD heart disease and possibly other disorders with membrane defect etiologies.

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Morpholino Oligomer–Mediated Exon Skipping Averts the Onset of Dystrophic Pathology in the mdx Mouse

Cited by 139 publications

References 41 publications

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges

Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs

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