Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy

2018 ◽  
Vol 10 (437) ◽  
pp. eaan0713 ◽  
Author(s):  
Hirofumi Komaki ◽  
Tetsuya Nagata ◽  
Takashi Saito ◽  
Satoru Masuda ◽  
Eri Takeshita ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Muscle Protein ◽  
Phase 1 ◽  
Exon Skipping ◽  
Muscle Disease ◽  
Dependent Manner ◽  
Reading Frame ◽  
Dystrophin Expression

Duchenne muscular dystrophy (DMD) is a lethal hereditary muscle disease caused by mutations in the gene encoding the muscle protein dystrophin. These mutations result in a shift in the open reading frame leading to loss of the dystrophin protein. Antisense oligonucleotides (ASOs) that induce exon skipping correct this frame shift during pre-mRNA splicing and partially restore dystrophin expression in mouse and dog models. We conducted a phase 1, open-label, dose-escalation clinical trial to determine the safety, pharmacokinetics, and activity of NS-065/NCNP-01, a morpholino ASO that enables skipping of exon 53. Ten patients with DMD (6 to 16 years old), carrying mutations in the dystrophin gene whose reading frame would be restored by exon 53 skipping, were administered NS-065/NCNP-01 at doses of 1.25, 5, or 20 mg/kg weekly for 12 weeks. The primary endpoint was safety; the secondary endpoints were pharmacokinetics and successful exon skipping. No severe adverse drug reactions were observed, and no treatment discontinuation occurred. Muscle biopsy samples were taken before and after treatment and compared by reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence, and Western blotting to assess the amount of exon 53 skipping and dystrophin expression. NS-065/NCNP-01 induced exon 53 skipping in dystrophin-encoding mRNA in a dose-dependent manner and increased the dystrophin/spectrin ratio in 7 of 10 patients. Furthermore, the amount of exon skipping correlated with the maximum drug concentration in plasma (Cmax) and the area under the concentration-time curve in plasma (AUC0-t). These results indicate that NS-065/NCNP-01 has a favorable safety profile and promising pharmacokinetics warranting further study in a phase 2 clinical trial.

scholarly journals Evaluating the potential of novel genetic approaches for the treatment of Duchenne muscular dystrophy

2021 ◽  
Author(s):  
Vratko Himič ◽  
Kay E. Davies
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Structural Integrity ◽  
Viral Vector ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Pharmacological Treatments ◽  
Genetic Sequencing ◽  
Reading Frame ◽  
Target Effects

AbstractDuchenne muscular dystrophy (DMD) is an X-linked progressive muscle-wasting disorder that is caused by a lack of functional dystrophin, a cytoplasmic protein necessary for the structural integrity of muscle. As variants in the dystrophin gene lead to a disruption of the reading frame, pharmacological treatments have only limited efficacy; there is currently no effective therapy and consequently, a significant unmet clinical need for DMD. Recently, novel genetic approaches have shown real promise in treating DMD, with advancements in the efficacy and tropism of exon skipping and surrogate gene therapy. CRISPR-Cas9 has the potential to be a ‘one-hit’ curative treatment in the coming decade. The current limitations of gene editing, such as off-target effects and immunogenicity, are in fact partly constraints of the delivery method itself, and thus research focus has shifted to improving the viral vector. In order to halt the loss of ambulation, early diagnosis and treatment will be pivotal. In an era where genetic sequencing is increasingly utilised in the clinic, genetic therapies will play a progressively central role in DMD therapy. This review delineates the relative merits of cutting-edge genetic approaches, as well as the challenges that still need to be overcome before they become clinically viable.

scholarly journals The Interplay of Mitophagy and Inflammation in Duchenne Muscular Dystrophy

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 648
Author(s):  
Andrea L. Reid ◽  
Matthew S. Alexander
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mitochondrial Dysfunction ◽  
Disease Onset ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Muscle Disease ◽  
Mitochondrial Morphology ◽  
Gene Therapies ◽  
Dystrophin Protein

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a pathogenic disruption of the DYSTROPHIN gene that results in non-functional dystrophin protein. DMD patients experience loss of ambulation, cardiac arrhythmia, metabolic syndrome, and respiratory failure. At the molecular level, the lack of dystrophin in the muscle results in myofiber death, fibrotic infiltration, and mitochondrial dysfunction. There is no cure for DMD, although dystrophin-replacement gene therapies and exon-skipping approaches are being pursued in clinical trials. Mitochondrial dysfunction is one of the first cellular changes seen in DMD myofibers, occurring prior to muscle disease onset and progresses with disease severity. This is seen by reduced mitochondrial function, abnormal mitochondrial morphology and impaired mitophagy (degradation of damaged mitochondria). Dysfunctional mitochondria release high levels of reactive oxygen species (ROS), which can activate pro-inflammatory pathways such as IL-1β and IL-6. Impaired mitophagy in DMD results in increased inflammation and further aggravates disease pathology, evidenced by increased muscle damage and increased fibrosis. This review will focus on the critical interplay between mitophagy and inflammation in Duchenne muscular dystrophy as a pathological mechanism, as well as describe both candidate and established therapeutic targets that regulate these pathways.

Eteplirsen treatment for Duchenne muscular dystrophy

Neurology ◽  
2018 ◽  
Vol 90 (24) ◽  
pp. e2146-e2154 ◽  
Author(s):  
Jay S. Charleston ◽  
Frederick J. Schnell ◽  
Johannes Dworzak ◽  
Cas Donoghue ◽  
Sarah Lewis ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Western Blot ◽  
Reverse Transcription ◽  
Exon Skipping ◽  
Fold Increase ◽  
Cell Penetration ◽  
Reverse Transcription Pcr ◽  
Dystrophin Expression ◽  
Immunofluorescence Intensity

ObjectiveTo describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen.MethodsClinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen.ResultsReverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001).ConclusionTaken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression.Classification of evidenceThis study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

scholarly journals Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients

2013 ◽  
Vol 21 (9) ◽  
pp. 1718-1726 ◽  
Author(s):  
David G Ousterout ◽  
Pablo Perez-Pinera ◽  
Pratiksha I Thakore ◽  
Ami M Kabadi ◽  
Matthew T Brown ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genome Editing ◽  
Reading Frame ◽  
Dystrophin Expression ◽  
In Cells

scholarly journals Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches

2019 ◽  
Vol 9 (1) ◽  
pp. 1 ◽  
Author(s):  
Yuko Shimizu-Motohashi ◽  
Hirofumi Komaki ◽  
Norio Motohashi ◽  
Shin’ichi Takeda ◽  
Toshifumi Yokota ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Stop Codon ◽  
Genetic Disorder ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Current Status ◽  
Therapeutic Approaches ◽  
Dystrophin Expression

Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by progressive muscle weakness, is caused by the absence or a decreased amount of the muscle cytoskeletal protein dystrophin. Currently, several therapeutic approaches to cure DMD are being investigated, which can be categorized into two groups: therapies that aim to restore dystrophin expression, and those that aim to compensate for the lack of dystrophin. Therapies that restore dystrophin expression include read-through therapy, exon skipping, vector-mediated gene therapy, and cell therapy. Of these approaches, the most advanced are the read-through and exon skipping therapies. In 2014, ataluren, a drug that can promote ribosomal read-through of mRNA containing a premature stop codon, was conditionally approved in Europe. In 2016, eteplirsen, a morpholino-based chemical capable of skipping exon 51 in premature mRNA, received conditional approval in the USA. Clinical trials on vector-mediated gene therapy carrying micro- and mini- dystrophin are underway. More innovative therapeutic approaches include CRISPR/Cas9-based genome editing and stem cell-based cell therapies. Here we review the current status of therapeutic approaches for DMD, focusing on therapeutic approaches that can restore dystrophin.

WED 237 Phosphorodiamidate morpholino oligomers for treatment of duchenne muscular dystrophy

2018 ◽  
Vol 89 (10) ◽  
pp. A34.2-A34
Author(s):  
Maresh Kate ◽  
Tiet May ◽  
Guglieri Michela ◽  
Domingos Joana ◽  
Straub Volker ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Western Blot ◽  
Clinical Studies ◽  
Novel Mutation ◽  
Cumulative Risk ◽  
Exon Skipping ◽  
Dystrophin Expression ◽  
Polymerase Chain ◽  
Phosphorodiamidate Morpholino Oligomers

Exon skipping is a novel, mutation-specific approach to treating patients with Duchenne muscular dystrophy (DMD). Phosphorodiamidate morpholino oligomers are nucleic acid analogues that selectively redirect pre-mRNA splicing to enable production of internally truncated dystrophin.In exon 51 skipping (eteplirsen; n=36) and exon 53 skipping (golodirsen; n=25) clinical studies, internally shortened dystrophin mRNA was observed in all treated patients (per reverse transcription polymerase chain reaction). Eteplirsen increased dystrophin expression 15.5-fold, 11.6-fold, and 2.4-fold vs untreated controls (percent dystrophin-positive fibres, Western blot, and immunohistochemistry intensity, respectively; all, p≤0.007) in a 180 week study, and 2.8-fold (Western blot; p=0.008) in a 48 week study. Golodirsen increased dystrophin expression 10.7-fold (Western blot) over baseline following 48 weeks of treatment. Over 4 years, versus comparable external controls, eteplirsen slowed ambulatory decline (6 min walk test difference, 165 m; p=0.001) and cumulative risk of losing ambulation (83% vs 17%). In 2 clinical studies that included non-ambulatory patients, eteplirsen slowed pulmonary decline versus natural history data (assessed by spirometry).Eteplirsen and golodirsen demonstrated clinical and biochemical effects in patients with DMD; ongoing studies of these compounds are further characterising their effects in various patient populations.

scholarly journals Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing

2021 ◽  
Vol 7 (18) ◽  
pp. eabg4910
Author(s):  
F. Chemello ◽  
A. C. Chai ◽  
H. Li ◽  
C. Rodriguez-Caycedo ◽  
E. Sanchez-Ortiz ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Membrane Integrity ◽  
Dystrophin Gene ◽  
Muscle Disease ◽  
Muscle Membrane ◽  
Reading Frame ◽  
Split Intein ◽  
Virus Serotype

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.

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