Exon Skipping and Duchenne Muscular Dystrophy Therapy: Selection of the Most Active U1 snRNA Antisense Able to Induce Dystrophin Exon 51 Skipping

One promising approach for the gene therapy of Duchenne muscular dystrophy (DMD) is exon skipping. When thinking of possible intervention on human, it is very crucial to identify the most appropriate antisense sequences able to provide the highest possible skipping efficiency. In this article, we compared the exon 51 skipping activity of 10 different antisense molecules, raised against splice junctions and/or exonic splicing enhancers (ESEs), expressed as part of the U1 small nuclear RNA (snRNA). The effectiveness of each construct was tested in human DMD myoblasts carrying the deletion of exons 48–50, which can be treated with skipping of exon 51. Our results show that the highest skipping activity and dystrophin rescue is achieved upon expression of a U1 snRNA-derived antisense molecule targeting exon 51 splice sites in combination with an internal exon sequence. The efficacy of this molecule was further proven on an exon 45–50 deletion background, utilizing patient''s fibroblasts transdifferentiated into myoblasts. In this system, we showed that the selected antisense was able to produce 50% skipping of exon 51.     

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

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Correction of Three Prominent Mutations in Mouse and Human Models of Duchenne Muscular Dystrophy by Single-Cut Genome Editing

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Direct Delivery of Antisense Oligonucleotides to the Middle and Inner Ear Improves Hearing and Balance in Usher Mice

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PTEN Inhibition Ameliorates Muscle Degeneration and Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy

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Application of 3-D Echocardiography and Gated Micro-Computed Tomography to Assess Cardiomyopathy in a Mouse Model of Duchenne Muscular Dystrophy

The purpose of this study was to measure changes in cardiac function as cardiomyopathy progresses in a mouse model of Duchenne muscular dystrophy using 3-D ECG-gated echocardiography. This study is the first to correlate cardiac volumes acquired using 3-D echocardiography with those acquired using retrospectively gated micro-computed tomography (CT). Both were further compared with standard M-mode echocardiography and histologic analyses. We found that although each modality measures a decrease in cardiac function as disease progresses in mdx/utrn^–/– mice (n = 5) compared with healthy C57BL/6 mice (n = 8), 3-D echocardiography has higher agreement with gold-standard measurements acquired by gated micro-CT, with little standard deviation between measurements. M-Mode echocardiography measurements, in comparison, exhibit considerably greater variability and user bias. Given the radiation dose associated with micro-CT and the geometric assumptions made in M-mode echocardiography to calculate ventricular volume, we suggest that use of 3-D echocardiography has important advantages that may allow for the measurement of early disease changes that occur before overt cardiomyopathy.     

Successful Regional Delivery and Long-term Expression of a Dystrophin Gene in Canine Muscular Dystrophy: A Preclinical Model for Human Therapies

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.