Restoring dystrophin expression in duchenne muscular dystrophy muscle progress in exon skipping and stop codon read through

The identification of the Duchenne muscular dystrophy gene and protein in the late 1980s led to high hopes of rapid translation to molecular therapeutics. These hopes were fueled by early reports of delivering new functional genes to dystrophic muscle in mouse models using gene therapy and stem cell transplantation. However, significant barriers have thwarted translation of these approaches to true therapies, including insufficient therapeutic material (eg, cells and viral vectors), challenges in systemic delivery, and immunological hurdles. An alternative approach is to repair the patient's own gene. Two innovative small-molecule approaches have emerged as front-line molecular therapeutics: exon skipping and stop codon read through. Both approaches are in human clinical trials and aim to coax dystrophin protein production from otherwise inactive mutant genes. In the clinically severe dog model of Duchenne muscular dystrophy, the exon-skipping approach recently improved multiple functional outcomes. We discuss the status of these two methods aimed at inducing de novo dystrophin production from mutant genes and review implications for other disorders.     

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.     

DMD患者骨骼肌抗肌萎缩蛋白表达与临床病理改变

目的探讨抗肌萎缩蛋白（dystrophin）免疫组织化学检查的临床价值及与Duchenne型肌营养不良（DMD）临床病理改变之问的相关性。方法通过组织学观察和免疫组织化学方法，对36例DMD患者骨骼肌dystrophin的表达情况、临床表现和肌肉病理改变进行观察分析。结果发现25例年龄在4岁以上的患儿多有比较典型的DMD临床表现，而11例4岁以下患儿症状比较轻。肌肉病理显示15例早期改变，17例中期改变，4例晚期改变，病理改变的严重程度与年龄相关。免疫组化染色显示36例患者的肌肉标本均有严重的dystrophin缺失，其中9例完全缺失，10例部分肌纤维膜有微弱着色，17例极少数肌纤维膜清楚着色，dystrophin的表达分级与病理改变分期及年龄无明显相关。结论检查dystrophin在肌纤维膜上的表达对DMD具有特异性诊断价值，但临床病理改变的严重程度主要与年龄和病程有关。     

Erratum: A novel splice site mutation (3157+1G>T) in the dystrophin gene causing total exon skipping and DMD phenotype

Erratum: An error was printed in the original version of this article in the Comments section, paragraph 2, relating to the size of exon 22 and the RT‐PCR product size described as resulting from the mutation 3157+1G>T. The paragraph should read: “We report a case of a 5 year old DMD patient with a novel splice site mutation affecting the GT dinucleotide splice donor of exon 22. The RT‐PCR analysis with primer sets spanning dystrophin exons 17‐25 amplified no normal size fragment (1251 bp), but a product shorter by 146 bp (the length of exon 22). Direct sequencing of the faster migrating fragment revealed total skipping of exon 22.”     

Rescue of severely affected dystrophin/utrophin-deficient mice through scAAV-U7snRNA-mediated exon skipping

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.     

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.     

Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy

Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD.     

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.     

Novel synonymous substitution in POMGNT1 promotes exon skipping in a patient with congenital muscular dystrophy

Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, congenital muscular dystrophy type 1C, and congenital muscular dystrophy type 1D are overlapping clinical entities belonging to a subgroup of the congenital muscular dystrophies (CMD), collectively designated dystroglycanopathies, in which the common underlying defect is hypoglycosylation of alfa-dystroglycan. Currently, six different genes are known to be implicated in these diseases: POMT1, POMT2, POMGNT1, FCMD, FKRP, and LARGE. We report the molecular characterization of a patient presenting clinical features of CMD and reduced immunostaining for alfa-dystroglycan in muscle. Three candidate genes (FCMD, POMT1 and POMGNT1) were analyzed, and a total of 18 sequence variants were detected: 15 polymorphisms in POMT1 [including three unreported single nucleotide polymorphisms (SNPs)], two polymorphisms in FCMD, and the exonic silent mutation c.636C > T in POMGNT1. Expression analysis revealed that this apparently silent mutation compromises correct premessenger RNA (mRNA) splicing, promoting skipping of the entire exon 7, with a consequent frameshift. In silico analysis of this mutation did not predict alterations in the canonical splice sequences, but rather the creation of a new exonic splice silencer. The recognition of such disease-causing elements may contribute to the further understanding of RNA processing and assist mutation screening in routine diagnosis, where such changes may be underestimated. To aid clinical diagnosis, we generated publicly available LOVD-powered Locus Specific Databases for these three genes and recorded all known sequence variants ( http://www.dmd.nl ).     

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.