Dataset Information

Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

ABSTRACT: Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45-55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45-55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45-55 skipping by intravenous injections of 6?mg/kg 10-Vivo-Morpholino cocktail (0.6?mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45-55 region was induced, and the Western blot analysis exhibited the restoration of 5-27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model.

SUBMITTER: Echigoya Y

PROVIDER: S-EPMC4345310 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

Echigoya Yusuke Y Aoki Yoshitsugu Y Miskew Bailey B Panesar Dharminder D Touznik Aleksander A Nagata Tetsuya T Tanihata Jun J Nakamura Akinori A Nagaraju Kanneboyina K Yokota Toshifumi T

Molecular therapy. Nucleic acids 20150203

Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45-55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45-55 skipping with a cocktail of Vivo-Morpholinos in ...[more]

PMID: 25647512

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Project description:Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45-55 skipping in dystrophin is potentially applicable to approximately 47% of DMD patients because many mutations occur within this "mutation hotspot." In addition, patients naturally harboring a dystrophin exons 45-55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. As such, exons 45-55 skipping could transform the DMD phenotype into an asymptomatic or very mild BMD phenotype and rescue nearly a half of DMD patients. In addition, this strategy is potentially applicable to some BMD patients as well, who have in-frame deletion mutations in this region. As the degree of exon skipping correlates with therapeutic outcomes, reliable measurements of exon skipping efficiencies are essential to the development of novel antisense-mediated exon skipping therapeutics. In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45-55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.

Project description:BackgroundBecker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype.MethodsWe use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography.ResultsOverall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes.ConclusionsThe bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Dataset Information

Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

Publications

Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

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