Characterization of a complex Duchenne muscular dystrophy-causing dystrophin gene inversion and restoration of the reading frame by induced exon skipping

Out of three mutations in the dystrophin gene that cause Duchenne muscular dystrophy (DMD), the most common, serious childhood muscle wasting disease, two are genomic deletions of one or more exons that disrupt the reading frame. Specific removal of an exon flanking a genomic deletion using antisense oligonucleotide intervention during pre-RNA processing can restore the reading frame and could potentially reduce disease severity. We describe a rare dystrophin gene rearrangement; inversion of approximately 28 kb, flanked by a 10-bp duplication and an 11-kb deletion, which led to the omission of exons 49 and 50 from the mature mRNA and the variable inclusion of several pseudoexons. In vitro transfection of cultured patient cells with antisense oligonucleotides directed at exon 51 induced efficient removal of that exon, as well as one of the more commonly included pseudoexons, suggesting closely coordinated splicing of these exons. Surprisingly, several antisense oligonucleotides (AOs) directed at this pseudoexon had no detectable effect on the splicing pattern, while all AOs directed at the other predominant pseudoexon efficiently excised that target. Antisense oligomers targeting dystrophin exon 51 for removal are currently undergoing clinical trials. Despite the unique nature of the dystrophin gene rearrangement described here, a personalized multiexon skipping treatment is applicable and includes one compound entering clinical trials for DMD.     

Antisense oligonucleotide-mediated exon skipping for Duchenne muscular dystrophy: progress and challenges

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 preclinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.     

Long-term rescue of dystrophin expression and improvement in muscle pathology and function in dystrophic mdx mice by peptide-conjugated morpholino

Exon skipping is capable of correcting frameshift and nonsense mutations in Duchenne muscular dystrophy. Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of Duchenne muscular dystrophy patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2'-O-methyl phosphorothioate. Peptide-conjugated phosphorodiamidate morpholino offers significantly higher efficiency than phosphorodiamidate morpholino, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of peptide-conjugated phosphorodiamidate morpholino targeting exon 23 in dystrophic mdx mice. The LD(50) of peptide-conjugated phosphorodiamidate morpholino was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg peptide-conjugated phosphorodiamidate morpholino produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg peptide-conjugated phosphorodiamidate morpholino restored dystrophin to >50% normal levels in skeletal muscle, and 15% in cardiac muscle. This was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of peptide-conjugated phosphorodiamidate morpholino can be safely applied to achieve significant therapeutic effects in an animal model.     

Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice

Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor–beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.     

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded ‘revertant’ dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Suppression of revertant fibers in mdx mice by expression of a functional dystrophin.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration that results from the absence of dystrophin. Despite null mutations in the dystrophin gene, many DMD patients display a low percentage of dystrophin-positive fibers. These "revertant fibers" are also present in the dystrophin-deficient mdx mouse and are believed to result from alternative splicing or second mutation events that bypass the mutation and restore an open reading frame. However, it is unclear what role dystrophin and the dystrophic pathology might play in revertant fiber formation and accumulation. We have analyzed the role of dystrophin expression and the dystrophic pathology in this process by monitoring revertant fibers in transgenic mdx mice that express truncated dystrophins. We found that newborn transgenic mice displayed approximately the same number of revertant fibers as newborn mdx mice, indicating that expression of a functional dystrophin does not suppress the initiation of revertant fiber formation. Surprisingly, when the transgene encoded a functional dystrophin, revertant fibers were not detected in adult or old mdx mice. In contrast, adult transgenic mice expressing a non-functional dystrophin accumulated increasing numbers of revertant fibers, similar to mdx mice, suggesting that positive selection is required for the persistence of revertant fibers. Finally, we provide evidence that the loss of revertant dystrophin in transgenic mdx muscle fibers overexpressing a functional dystrophin results from displacement of the revertant protein by the transgene-encoded dystrophin.     

Efficient bypass of mutations in dysferlin deficient patient cells by antisense‐induced exon skipping

Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 2B and Miyoshi myopathy. More than 350 different sequence variants have been reported in DYSF. Like dystrophin, the size of the dysferlin mRNA is above the limited packaging size of AAV vectors. Alternative strategies to AAV gene transfer in muscle cells must then be addressed for patients. A gene therapy approach for Duchenne muscular dystrophy was recently developed, based on exon‐skipping strategy. Numerous sequences are recognized by splicing protein complexes and, when specifically blocked by antisense oligoucleotides (AON), the corresponding exon is skipped. We hypothesized that this approach could be useful for patients affected with dysferlinopathies. To confirm this assumption, exon 32 was selected as a prioritary target for exon skipping strategy. This option was initially driven by the report from Sinnreich and colleagues of a patient with a very mild and late‐onset phenotype associated to a natural skipping of exon 32. Three different antisense oligonucleotides were tested in myoblasts generated from control and patient MyoD transduced fibroblasts, either as oligonucleotides or after incorporation into lentiviral vectors. These approaches led to a high efficiency of exon 32 skipping. Therefore, these results seem promising, and could be applied to several other exons in the DYSF gene. Patients carrying mutations in exons whose the in‐frame suppression has been proven to have no major consequences on the protein function, might benefit of exon‐skipping based gene correction. Hum Mutat 30:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Muscle Plasma Membrane Changes in Dystrophin Gene Exon 52 Knockout Mouse

Changes in muscle plasma membranes in mice lacking exon 52 of the dystrophin gene (mdx52 mouse) were studied using the freeze-fracture technique. The extensor digitorum longus (EDL) muscle plasma membrane of the mdx52 mouse at 8 weeks of age showed significantly increased caveola density (p < 0.05 by two-tailed t-test) and significantly decreased densities of intramembranous particles (IMPs), orthogonal arrays (OAs) and orthogonal array subunit particles (OASPs) (p < 0.05 by two-tailed t-test, p < 0.01 by Wilcoxon rank-sum test, p < 0.05 by two-tailed t-test, respectively) on the protoplasmic face when compared with those of control EDL muscles. These changes are more similar to those seen in DMD than those in the mdx mouse at the same age as reported previously. Thus, the gene abnormality in the different exon of the mouse dystrophin gene seems to induce somewhat different changes in the muscle plasma membrane.     

An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements

A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific binding site (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer in the central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifs that collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowing the occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.     

Autoimmune response and its long-term consequences after exon-skipping therapy in a Duchenne muscular dystrophy mouse model

The progress of antisense-based therapies using first generation Morpholino oligonucleotides for Duchenne muscular dystrophy (DMD) is expected to partially restore dystrophin expression and may prolong the lifespan of DMD patients. In a recent issue of The Journal of Pathology, a sophisticated study by Vila et al used a dystrophic mouse model of DMD to demonstrate that Morpholino-induced exon skipping induced dystrophin expression in skeletal muscle and stimulated cell mediated and humoral responses to dystrophin. The study highlights the need to further investigate the autoimmune response against de novo synthesised truncated dystrophin protein and its long-term consequences after exon-skipping therapy for DMD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.