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Duchenne muscular dystrophy is a fatal neuromuscular disorder affecting around one in 3,500-5,000 male births that is characterized by progressive muscular deterioration. It is inherited in an X-linked recessive fashion and is caused by loss-of-function mutations in the gene coding for dystrophin, a cytoskeletal protein that stabilizes the plasma membrane of muscle fibers. In September 2016, the US Food and Drug Administration granted accelerated approval for eteplirsen (or Exondys 51), a drug that acts to promote dystrophin production by restoring the translational reading frame of through specific skipping of exon 51 in defective gene variants. Eteplirsen is applicable for approximately 14% of patients with mutations. This article extensively reviews and discusses the available information on eteplirsen to date, focusing on pharmacological, efficacy, safety, and tolerability data from preclinical and clinical trials. Issues faced by eteplirsen, particularly those relating to its efficacy, will be identified. Finally, the place of eteplirsen and exon skipping as a general therapeutic strategy in Duchenne muscular dystrophy treatment will be discussed.

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55–skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55–skipping DMD therapy.

Duchenne muscular dystrophy (DMD), a fatal X-linked recessive disorder, is caused mostly by frame-disrupting, out-of-frame deletions in the dystrophin (DMD) gene. Antisense oligonucleotide-mediated exon skipping is a promising therapy for DMD. Exon skipping aims to convert out-of-frame mRNA to in-frame mRNA and induce the production of internally-deleted dystrophin as seen in the less severe Becker muscular dystrophy. Currently, multiple exon skipping has gained special interest as a new therapeutic modality for this approach. Previous retrospective database studies represented a potential therapeutic application of multiple exon skipping. Since then, public DMD databases have become more useful with an increase in patient registration and advances in molecular diagnosis. Here, we provide an update on DMD genotype-phenotype associations using a global DMD database and further provide the rationale for multiple exon skipping development, particularly for exons 45–55 skipping and an emerging therapeutic concept, exons 3–9 skipping. Importantly, this review highlights the potential of multiple exon skipping for enabling the production of functionally-corrected dystrophin and for treating symptomatic patients not only with out-of-frame deletions but also those with in-frame deletions. We will also discuss prospects and challenges in multiple exon skipping therapy, referring to recent progress in antisense chemistry and design, as well as disease models.

The most common neuromuscular disorder, Duchenne muscular dystrophy (DMD), is caused by mutations in the DMD gene, resulting in a lack of dystrophin. In addition to severe and progressive muscle wasting, a subset of individuals with DMD experience, to largely varying extents, behavioural and cognitive deficits, including a lower IQ, and neurological comorbidities, such as autism spectrum disorder, obsessive compulsive disorder and attention deficit hyperactivity disorder. Neuroimaging studies in individuals with DMD have identified widespread pathology, including structural, physiological and connective alterations. DMD mouse models exhibit a number of DMD-associated behavioural traits, including anxiety, social deficits and learning disabilities, and have been used to investigate DMD brain pathology. Although there are currently no therapies to treat DMD brain pathology, genetic approaches are being developed to restore dystrophin expression. In particular, the exon skipping approach shows promise in ameliorating certain DMD-associated behavioural deficits in preclinical settings. However, the therapeutic potential of postnatal restoration of dystrophin isoforms involved in neurodevelopment is unknown. Furthermore, challenges such as low dystrophin restoration efficacy and translatability from DMD mouse models to the clinic remain to be addressed.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

Background Alport syndrome (AS) is a genetic kidney disease caused by a mutation in type IV collagen α3, α4, and α5, which are normally secreted as heterotrimer α345(IV). Nonsense mutation in these genes causes severe AS phenotype. We previously revealed that the exon-skipping approach to remove a nonsense mutation in α5(IV) ameliorated the AS pathology. However, the effect of removing an exon on trimerization is unknown. Here, we assessed the impact of exon deletion on trimerization to evaluate their possible therapeutic applicability and to predict the severity of mutations associated with exon-skipping. Methods We produced exon deletion constructs (ΔExon), nonsense, and missense mutants by mutagenesis and evaluated their trimer formation and secretion activities using a nanoluciferase-based assay that we previously developed. Results Exon-skipping had differential effects on the trimer secretion of α345(IV). Some ΔExons could form and secrete α345(IV) trimers and had higher activity compared with nonsense mutants. Other ΔExons had low secretion activity, especially for those with exon deletion near the C-terminal end although the intracellular trimerization was normal. No difference was noted in the secretion of missense mutants and their ΔExon counterpart. Conclusion Exon skipping is advantageous for nonsense mutants in AS with severe phenotypes and early onset of renal failure but applications may be limited to ΔExons capable of normal trimerization and secretion. This study provides information on α5(IV) exon-skipping for possible therapeutic application and the prediction of the trimer behavior associated with exon-skipping in Alport syndrome.

Antisense oligonucleotide (AO)‐mediated exon‐skipping therapies show promise in Duchenne muscular dystrophy (DMD), a devastating muscular disease caused by frame‐disrupting mutations in the DMD gene. However, insufficient systemic delivery remains a hurdle to clinical deployment. Here, we demonstrate that MOTS‐c, a mitochondria‐derived bioactive peptide, with an intrinsic muscle‐targeting property, augmented glycolytic flux and energy production capacity of dystrophic muscles in vitro and in vivo , resulting in enhanced phosphorodiamidate morpholino oligomer (PMO) uptake and activity in mdx mice. Long‐term repeated administration of MOTS‐c (500 μg) and PMO at the dose of 12.5 mg/kg/week for 3 weeks followed by 12.5 mg/kg/month for 3 months (PMO‐M) induced therapeutic levels of dystrophin expression in peripheral muscles, with up to 25‐fold increase in diaphragm of mdx mice over PMO alone. PMO‐M improved muscle function and pathologies in mdx mice without detectable toxicity. Our results demonstrate that MOTS‐c enables enhanced PMO uptake and activity in dystrophic muscles by providing energy and may have therapeutic implications for exon‐skipping therapeutics in DMD and other energy‐deficient disorders. Synopsis This study demonstrates the use of MOTS‐c peptide to promote oligonucleotides uptake in muscle cells by augmenting glycolytic flux and energy production. This approach may be used to improve the success of DMD exon‐skipping therapy and potentially be used for other diseases with hallmarks of energy deficiency. The MOTS‐c resulted in increased ATP levels in dystrophic muscles. Co‐administration of MOTS‐c and low doses of PMO induced 25‐fold increase of dystrophin expression in dystrophic mice. PMO with MOTS‐c improved muscle function and pathologies in dystrophic mice. Graphical Abstract This study demonstrates the use of MOTS‐c peptide to promote oligonucleotides uptake in muscle cells by augmenting glycolytic flux and energy production. This approach may be used to improve the success of DMD exon‐skipping therapy and potentially be used for other diseases with hallmarks of energy deficiency.

Exon skipping is an effective strategy for the treatment of many Duchenne Muscular Dystrophy (DMD) mutations. Natural exon skipping observed in several DMD cases can help in identifying novel therapeutic tools. Here, we show a DMD study case where the lack of a splicing factor (Celf2a), which results in exon skipping and dystrophin rescue, is due to a maternally inherited trans‐generational epigenetic silencing. We found that the study case and his mother express a repressive long non‐coding RNA, DUXAP8, whose presence correlates with silencing of the Celf2a coding region. We also demonstrate that DUXAP8 expression is lost upon cell reprogramming and that, upon induction of iPSCs into myoblasts, Celf2a expression is recovered leading to the loss of exon skipping and loss of dystrophin synthesis. Finally, CRISPR/Cas9 inactivation of the splicing factor Celf2a was proven to ameliorate the pathological state in other DMD backgrounds establishing Celf2a ablation or inactivation as a novel therapeutic approach for the treatment of Duchenne Muscular Dystrophy. Synopsis The study shows that the inhibition of Celf2a splicing factor isoform is able to partially restore dystrophin production in a subgroup of patients affected by Duchenne Muscular Dystrophy (DMD). Moreover, Celf2a expression is epigenetically regulated and this regulation is mediated by a lncRNA. Partial dystrophin restoration in a DMDΔ44 genetic background was achieved by the depletion of the Celf2a splicing factor isoform. Celf2a restoration was obtained by reprogramming patient fibroblasts into iPSCs. Celf2a silencing, in the case study analysed, is due to a transgenerationally inherited epigenetic regulation. Celf2a regulation is mediated by the lncRNA DUXAP8. Graphical Abstract The study shows that the inhibition of Celf2a splicing factor isoform is able to partially restore dystrophin production in a subgroup of patients affected by Duchenne Muscular Dystrophy (DMD). Moreover, Celf2a expression is epigenetically regulated and this regulation is mediated by a lncRNA.

Introduction: Severity and disease progression in people with Cystic Fibrosis (CF) is typically dependent on their genotype. One potential therapeutic strategy for people with specific mutations is exon skipping with antisense oligonucleotides (AO). CFTR exon 9 is an in-frame exon and hence the exclusion of this exon would excise only 31 amino acids but not alter the reading frame of the remaining mRNA. Splice mutations 1209 + 1 G > C and 1209 + 2 T > G were documented to cause CFTR exon 9 skipping and these variants were reported to manifest as a milder CF disease, therefore exon 9 skipping could be beneficial for people with class I mutations that affect exon 9 such as p.Trp401X. While the impact of exon 9 skipping on gene expression and cellular pathways can be studied in cells in vitro , trace amount of full-length normal or mutated material could confound the evaluation. To overcome this limitation, the impact of CFTR exon 9 skipping on disease phenotype and severity is more effectively evaluated in a small animal model. It was hypothesised that antisense oligonucleotide-mediated skipping this particular exon could result in a “mild mouse CF phenotype”. Methods: Cftr exon 9 deleted mice were generated using homologous recombination. Survival of homozygous ( Cftr Δ9/Δ9 ) and heterozygous ( Cftr Δ9/+ ) mice was compared to that of other CF mouse models, and lung and intestinal organ histology examined for any pathologies. Primary airway epithelial cells (pAECs) were harvested from Cftr Δ9/Δ9 mice and cultured at the Air Liquid Interface for CFTR functional assessment using Ussing Chamber analysis. Results: A Cftr Δ9/Δ9 mouse model presented with intestinal obstructions, and at time of weaning (21 days). Cftr Δ9/Δ9 mice had a survival rate of 83% that dropped to 38% by day 50. Histological sections of the small intestine from Cftr Δ9/Δ9 mice showed more goblet cells and mucus accumulation than samples from the Cftr Δ9/+ littermates. Airway epithelial cell cultures established from Cftr Δ9/Δ9 mice were not responsive to forskolin stimulation. Summary: The effect of Cftr exon 9 deletion on Cftr function was assessed and it was determined that the encoded Cftr isoform did not result in a milder “mouse CF disease phenotype,” suggesting that Cftr exon 9 is not dispensable, although further investigation in human CF pAECs would be required to confirm this observation.