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Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000–6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD. [ABSTRACT FROM AUTHOR]

DRIS is a 2 ′ - O -methyl-phosphorothioate antisense oligonucleotide designed to skip exon 51 in dystrophin pre-mRNA of boys with DMD. In a Ph 3 study (DMD114044), a 10 m treatment difference (NS) on 6-min walking distance (6MWD) in favour of DRIS was seen in the overall study population but there was a treatment benefit of 21 m in a pre-specified subset of boys aged ⩽7y ( n = 79), suggesting that younger, less progressed boys could benefit more from DRIS in terms of ambulatory endpoints vs older boys in whom DMD is more advanced. In an open-label extension (OLE; DMD114349) of two 48-wk placebo (PBO)-controlled trials (DMD114117, DMD114044), 96 wks’ DRIS resulted in a clinically meaningful difference from 48 wks’ PBO (feeder study) followed by DRIS (OLE) in 6MWD of 46 m ( n = 113; completed 48 wks’, DMD114349). By feeder study, the treatment effect was 52 m ( n = 30) for DMD114117 and 49 m for DMD114044 ( n = 83), for those on DRIS for 96 wks vs PBO/delayed treatment. Data suggest that treatment benefits that manifest early in less severe DMD are maintained, while in more advanced DMD, benefits may emerge only after prolonged exposure. In a pooled analysis of two comparable Ph 2 trials (DMD114117, DMD114876), a subset of boys with similar BL demographic and functional characteristics who received DRIS 6 mg/kg/wk ( n = 36) was compared with PBO-treated boys ( n = 34; Mixed-Effect Model Repeated Measure model including Treatment, Visit, Treatment by Visit, Country/Study, BL 6MWD, BL 6MWD by Visit). At Wk 24, there was a clinically meaningful and statistically significant difference in 6MWD between DRIS- and PBO-treated boys of 31 m ( n = 70; 95% confidence interval: 11, 51; p = 0.003), based on an adjusted mean (standard error) difference of −11(7) m for placebo and + 20(7) m for DRIS. DMD114044 enrolled boys aged ⩽16y, whereas in the pooled dataset, the oldest boys were only 13y, supporting the premise that treatment at a younger age is more immediately beneficial for preserving ambulatory capacity. [ABSTRACT FROM AUTHOR]

• This study contributes to the natural history of DMD, linking the ambulant and non-ambulant phases. • Respiratory measurements, upper limb function, pinch and grip force are reported. • A composite score combining respiratory outcomes, upper limb function and strength is explored. The field of translational research in Duchenne muscular dystrophy (DMD) has been transformed in the last decade by a number of therapeutic targets, mostly studied in ambulant patients. A paucity of studies focus on measures that capture the non-ambulant stage of the disease, and the transition between the ambulant and non-ambulant phase. In this prospective natural history study, we report the results of a comprehensive assessment of respiratory, upper limb function and upper limb muscle strength in a group of 89 DMD boys followed in 3 European countries, 81 receiving corticosteroids, spanning a wide age range (5–18 years) and functional abilities, from ambulant (n = 60) to non-ambulant (n = 29). Respiratory decline could be detected in the early ambulatory phase using Peak Expiratory Flow percentage predicted (PEF%), despite glucocorticoid use (mean annual decline: 4.08, 95% CI [−7.44,−0.72], p = 0.02 in ambulant; 4.81, 95% CI [−6.79,−2.82], p < 0.001 in non-ambulant). FVC% captured disease progression in non-ambulant DMD subjects, with an annual loss of 5.47% (95% CI [−6.48,−4.45], p < 0.001). Upper limb function measured with the Performance of Upper Limb (PUL 1.2) showed an annual loss of 4.13 points (95% CI [−4.79,3.47], p < 0.001) in the non-ambulant cohort. Measures of upper limb strength (MyoGrip and MyoPinch) showed a continuous decline independent of the ambulatory status, when reported as percentage predicted (grip force −5.51%, 95% CI [−6.54,−4.48], p < 0.001 in ambulant and a slower decline −2.86%; 95% CI −3.29,−2.43, p < 0.001, in non-ambulant; pinch force: −2.66%, 95% CI [−3.82,−1.51], p < 0.001 in ambulant and −2.23%, 95% CI [−2.92,−1.53], p < 0.001 in non-ambulant). Furthermore, we also explored the novel concept of a composite endpoint by combining respiratory, upper limb function and force domains: we were able to identify clear clinical progression in patients in whom an isolated measurement of only one of these domains failed to appreciate the yearly change. Our study contributes to the field of natural history of DMD, linking the ambulant and non-ambulant phases of the disease, and suggests that composite scores should be explored further. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is caused by a lack of dystrophin. Several strategies exist to restore dystrophin production, including exon skipping and gene therapy. However, dystrophin epitopes, expressed in revertant fibres in non-treated patients, may elicit a T cell-mediated immune response, which could reduce the efficacy of therapies designed to restore dystrophin. We performed enzyme-linked immunospot interferon-gamma assays on peripheral blood mononuclear cells from 78 paediatric patients with DMD from a natural history study. Immune responses to dystrophin were quantified using 368 peptides spanning the entire protein, organized into nine pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Overall, our results show that pre-existing T cell responses to dystrophin are uncommon (8%), inconsistent and low-level. Whilst this does provide some confidence for dystrophin restorative treatments, the fact that some patients are responsive warrants that individuals be screened prior to treatment. [ABSTRACT FROM AUTHOR]

Abstract: Upper limb assessment in non-ambulant patients remains a challenge. We have designed new tools to precisely assess pinch (MyoPinch), grip (MyoGrip), wrist flexion and extension (MyoWrist) strength. We have also designed a new tool to assess the ability of patients to produce repetitive flexion/extension movements of wrist and fingers (MoviPlate). We have assessed the feasibility and reliability of these new tools in 30 non-ambulant patients with Duchenne muscular dystrophy and in 30 age-matched male controls. Existing measures, such as Motor Function Measure, Tapping, and the Brooke Upper Extremity Functional Rating Scale were also performed. Results demonstrated that assessments were feasible in nearly all upper limbs tested for MyoGrip, MyoPinch and MoviPlate. The reliability of all tests, including MyoWrist which was not feasible in the patients presenting with contractures, was excellent in patients as in controls. Motor capacities decrease with the number of months spent in the wheelchair. The scores in the tests were partially correlated with each other, and with clinical measures such as vital capacity, Motor Function Measure, functional hand scale and Brooke score. This study validates a panel of upper limb muscle strength and function measures for Duchenne Muscular Dystrophy which can be applied from controls to extremely weak patients. [Copyright &y& Elsevier]

Adeno Associated virus serotype 8 (AAV8) is of particular interest as a vector for pre-clinical and clinical trial for Duchenne Muscular Dystrophy (DMD). In several cell lines, this vector has been shown to enter cells through clathrin-mediated endocytosis followed by a trafficking through the microtubule network in various endosomal compartments toward the nucleus. To efficiently transduce cells, AAV must undergo multiple levels of regulation in these cellular compartments. In DMD, dystrophin deficiency results in disturbed balance of cellular events i.e., fiber centronucleation, disorganized cytoskeleton, presence of fibrosis. We have recently described a loss of virion genomes from both dogs and mice models of DMD treated with therapeutic molecules vectorized in AAV. Indeed, the pathophysiological state of DMD muscle should impact on virions fate and subsequently affect crucial steps for AAV effectiveness as viral uncoating, viral genome maintenance and consequently, the transduction efficiency of AAV. Our project aims to characterize cellular uptake and intracellular transport of AAV8 in DMD muscular cells, with the goal of optimizing AAV vector use to get the best transduction efficiency with the lowest AAV dose. Our first data showed that AAV8-GFP was less efficient to transduce DMD and control primary muscular cells compared to HeLa cells. Moreover, AAV8 traffics through same endosomal compartment in DMD and control myoblasts, but at different rates during early time points of the transduction. These results suggest that in muscle cells, AAV8 uses different entry and trafficking pathways from those previously described in HeLa cells and that dystrophic cellular status could affect subcellular processing of the vector particles. We will specify the relationship between AAV8 vector entry, trafficking, uncoating, and transduction efficiency in vitro in primary myoblasts/myotubes of DMD patients and controls. [ABSTRACT FROM AUTHOR]

The Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. About 60% of the affected boys are treatable by exon skipping, i.e. the selective removal of exons flanking an out-of frame mutation in the dystrophin messenger, which results in shorter in-frame transcripts that are translated into functionally active dystrophin. Recombinant adeno-associated virus (rAAV) derived vectors carrying modified U7snRNA sequences are promising tools to allow an efficient exon skipping. In order to prepare a clinical trial in non-ambulant DMD patients using a rAAV8-U7 vector injected in the forearm, we injected in one forelimb a total of 21 Golden Retriever Muscular Dystrophy (GRMD) dogs to determine the therapeutic dose of the product and establish safety of the treatment. Results demonstrated high dystrophin expression levels (up to 80%) in muscles of dogs injected with 2.5E13vg/kg. Strength improvement and correction of histological, NMR imaging and spectroscopy indices were demonstrated in muscles with more than 40% dystrophin expression. Finally, since a proportion of DMD patients are not amenable to treatment by exon skipping, we also evaluated a therapy based on the transfer of a rAAV8 vector encoding a sequence-optimized microdystrophin (MD1), which could eventually benefit all DMD patients. Three GRMD dogs were injected in one forelimb with this vector, exactly as in the U7-GRMD dogs. Treated limbs of these dogs exhibited consistent MD1 expression, which correlated with local correction of the pathology and strength improvement. The complete results obtained during these two pre-clinical trials will be presented and a comparison of these two therapeutic approaches will be discussed. Supported by the Association Française contre les Myopathies, the UK Muscular Dystrophy Campaign and by Advanced Diagnostics for New Therapeutic Approaches, a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by OSEO. [Copyright &y& Elsevier]

A clinical trial programme to assess safety and efficacy of drisapersen (DRIS) in DMD comprises two Phase 2, one Phase 3 placebo (PBO)-controlled, (total N = 290) and two open-label extension (OLE) trials ( N = 233). The Phase 2 studies enrolled comparable populations with relatively mild disease (mean baseline [BL] 6-min walking distance [6MWD] ∼400 m). At Wk 25, DMD114117 (117; N = 53) showed a statistically significant and clinically meaningful treatment difference of +35 m ( p = 0.014) in 6MWD for 6 mg/kg/wk continuous DRIS treatment vs PBO (maintained at Wk 49 [+36 m; p = 0.051]); %-predicted 6MWD improved by 6% ( p < 0.05) at both time points. After 24 wks’ continuous 6 mg/kg/wk DRIS treatment in DMD114876 ( N = 51), a clinically meaningful treatment difference was seen in 6MWD (+27 m; p = 0.069); %-predicted 6MWD improved by 5% ( p = 0.051). The Phase 3 study DMD114044 (044; N = 186) enrolled a more severe population (mean BL 6MWD < 350 m). Boys received 6 mg/kg/wk continuous DRIS or PBO for 48 wks resulting in a non-statistically significant +10 m difference in favour of DRIS. At Wk 48, for the combined analysis of 117 and 044 (⩽7 y at study BL), the treatment difference in 6MWD was +24 m ( p = 0.048) for DRIS 6 mg/kg/wk vs. PBO. At 96 wks in the OLE fed by 117 and 044, the treatment difference ( n = 113) was +46 m from BL for continuous 6 mg/kg DRIS vs starting DRIS treatment after 48 wks. In the Phase 1/2 OLE DMD114673 ( n = 10; average age: 12.9 y), the mean change in 6MWD at Wk 177 (3.4 y) was −24.5 m (median +7.5 m) from OLE BL. Most common AEs with DRIS treatment were injection-site reactions, mild reversible proteinuria. Infrequent moderate to severe thrombocytopenia was also seen. The results of the trial programme are promising, as deterioration of muscle function is associated with the natural history of DMD in young boys. Overall, observed improvements or reductions in decline in 6MWD over 24–96 wks at this stage of the disease is indicative of an encouraging effect in reducing disease progression. [ABSTRACT FROM AUTHOR]

The pre-symptomatic induction of inflammatory cascades and invasion of muscle by immune cells in dystrophin deficient muscle contributes to the pathology of Duchenne muscular dystrophy (DMD). Traditional and RNA-based gene therapy approaches for DMD are progressing through clinical development. Whilst in one study a post-treatment reduction in inflammatory infiltrate after antisense oligonucleotide-mediated exon skipping has been reported; another study, on viral delivered mini-dystrophin, has shown evidence that dystrophin epitopes expressed in revertant fibres can elicit T cell production in untreated patients which may accelerate a post-treatment immune response. To extend this observation, we are performing annual ELISPOT IFN-gamma assays on patients recruited into a four year DMD natural history study. The study is recruiting 30 ambulant and 30 non-ambulant DMD boys with exon skippable deletions from three clinical centres. ELISPOT assays on all patients are performed with a full length dystrophin peptide set whilst patients with exon 51 or 53 skippable deletions are also assessed with peptides corresponding to unique epitopes generated by exon skipping. This allows us to assess pre-existing immunological responses to dystrophin epitopes in patients prior to inclusion in clinical trials, as well as the likelihood of a post-exon skipping immune response to newly-generated dystrophin protein. Here we present the results to date from patients recruited during the first year; we correlated data to factors such as age, ambulation status, steroid regime and DMD deletion. The majority of patients did not have significant T cell activity against the full length or unique epitope peptide sets; whilst in those that did (approximately 1/6), the activity was relatively low. These ’positive’ patients are presented and the responses mapped to specific dystrophin peptides. Our data provides a useful immunological baseline for future DMD clinical trials. [Copyright &y& Elsevier]

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. About 60% of the affected boys are treatable by exon skipping, i.e. the selective removal of exons flanking an out-of frame mutation in the dystrophin messenger, which results in shorter in-frame transcripts that are translated into functionally active dystrophin. Recombinant AAV-derived vectors carrying modified U7snRNA sequences are promising tools to allow an efficient exon skipping. In order to prepare a clinical trial in non-ambulant DMD patients using a rAAV8-U7 vector injected in the forearm, we injected in one forelimb a total of 21 Golden Retriever Muscular Dystrophy (GRMD) dogs. All muscles of the limbs were observed about 14weeks later for dystrophin expression and histological patterns Depending on the injected dose and the injection protocol, we showed low to high (up to 80%) dystrophin expression levels in muscles of the injected legs depending of the vector dose. Improvement of the histological pattern was assessed by histomorphological analysis of the immunological detection of dystrophin expression, sclerosis (immunopexoxydase against Collagen I), regeneration (developmental Myosin Heavy Chain), calcium accumulation, anisocytosis, macrophagic infiltration, and lymphocytic infiltration. The dystrophin expression was characterized by a diffuse pattern with single or small groups of positive fibers in most fascicles. A significant decrease of sclerosis in muscles exhibiting more than 60% of dystrophin positive fibers and an important decrease of regeneration (in muscles containing up to 30% of dystrophin), was observed. These results show the efficiency of the rAAV8-U7 strategy to restrict the natural degradation of the dystrophic muscle. Supported by the Association Française contre les Myopathies and Advanced Diagnostics for New Therapeutic Approaches, a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by OSEO. [ABSTRACT FROM AUTHOR]

Exon skipping therapy is an emerging approach in Duchenne Muscular Dystrophy (DMD). Antisense oligonucleotides that skip exon 51, 44, 45, and 53 are currently evaluated in clinical trials. Knowing the precise phenotype of potentially eligible patients is important for designing the clinical trials. It is generally believed that there are few if any phenotypic differences between these different groups of patients or between these patients and the general DMD population. In preparation of exon 53 skipping mediated by an AAV8 construct, we recruited 24 patients aged from 6 to 20years in an observational study of upper limb strength and function, using the Motor Function Measure (MFM), the Myogrip, the MyoPinch and the MoviPlate, three tools already validated in a large non ambulant DMD population. Data of 14 non ambulant DMD patients (13.8±2.7years) at inclusion were compared with 14 age and size-matched DMD controls (13.7±2.6years) with all types of mutation. DMD 53 patients scored significantly lower in the MFM (D3: 66±17% vs. 82±13% for DMD 53 vs. DMD control), had significant lower handgrip and key pinch strength on both hands. They had lost ambulation 13months earlier (Age: 105±20 vs. 118±19 p =0.04 for DMD 53 vs. DMD control). In order to rule out that this is due to a selection bias, we compared DMD 53 patients with other DMD patients carrying a deletion not involving the 45–55 region and looked at DMD patient cohort identified at the Cochin hospital’s routine diagnostic laboratory. We found that 91 DMD ex53 patients had lost ambulation at 108 ±15months, whilst other 400 non 45–55 DMD patients with a deletion had lost ambulation at 139±54months. Taken together, these preliminary data demonstrate that non-ambulant patients treatable by exon 53 skipping present a more severe phenotype than the general DMD population. This must be taken into account in the design of studies concerning this population. [ABSTRACT FROM AUTHOR]

In the context of future AAV-based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We followed AAV genome copy numbers after AAV-U7 delivery in the mdx dystrophic mouse. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx muscles within three weeks after intra-muscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon-skipping, displayed much lower titers of viral genomes, showing that sub-optimal dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment. [Copyright &y& Elsevier]

Non-invasive measurements and quantitative evaluation of the natural course of disease need to be established in anticipation of replacement therapy in Duchenne muscular dystrophy (DMD). A cohort of 23 DMD boys (6–18years, 14 non-ambulant), were enrolled specifically to characterize disease progression of upper limbs, to be targeted in future therapeutic trials. Prior studies in the GRMD canine model showed that Nuclear Magnetic Resonance imaging (NMRI) and phosphorous spectroscopy (NMRS) could provide quantitative indices which graded with therapeutic dose. A similar approach was implemented in patients, who were examined in a 3T-60cm Siemens Magnetom TRIO system, with the arm along the body. The two arms were examined in separate sessions, to be repeated yearly, comprising NMRI (measurement of T2 of muscle water, and of fat fraction by 3-point Dixon imaging) and NMRS of phosphate metabolites. Each item lasted ∼20min. Twelve indices were analysed: [–] 2 in NMRI: %fat signal (%F), muscle water T2 (deconvoluted from fat) [–] 10 in NMRS: 8 ratios of metabolites combining: ATP, phosphocreatine, phospho mono- and di-esters and two pools of inorganic phosphate: cytosolic (Pia) and an anomalous alkaline pool present in dystrophic muscle (Pib), and 2 indices for respective pH values. T2 was above laboratory norms in 15% of cases, %F exceeded 10% of signal in 70% of cases and correlated to patient age (R =.58, P <0.001), as did 7 of 8 metabolic ratios (R =.38 to .46, P <0.01). All NMRS ratios correlated strongly to %F, (R =.46 to .77, P <0.001), but not pH values. NMRI indices showed flexor muscles were more affected than extensor muscles. Quantitative NMRI and NMRS provided a variety of indices which were abnormal in forearms of DMD patients, covering a broad range of fat infiltration, and metabolic abnormalities which graded together and with patient age and could provide biomarkers of therapeutic efficacy. [Copyright &y& Elsevier]

Abstract: Duchenne (DMD) and Becker (BMD) muscular dystrophies are muscle-wasting diseases caused by mutations in the DMD gene encoding dystrophin. Usually, out-of-frame deletions give rise to DMD whereas in-frame deletions result in BMD. BMD patients exhibit a less severe disease because an abnormal but functional dystrophin is produced. This is the rationale for attempts to correct the reading frame by using an exon-skipping strategy. In order to apply this approach to a larger number of patients, a strategy of exons 45–55 multi-skipping has been proposed, because it would correct the mRNA reading frame in almost 75% of DMD patients with a deletion. The resulting dystrophin lacks part of the binding site for the neuronal nitric oxide synthase (nNOSμ), which normally binds to spectrin-like repeats 16 and 17 of dystrophin. Knowing that these domains are encoded by exons 42–45, we investigated the nNOSμ status in muscle biopsies from twelve BMD patients with deletions of exons 45–55. We found a wide spectrum of nNOSμ expression and localization. The strictly cytosolic mislocalization of nNOSμμ was associated with the more severe phenotypes. The cytosolic NO production correlated with both hypernitrosylation of the sarcoplasmic reticulum calcium-release-channel RyR1 and release of calstabin-1, a central hub to Ca2+ signaling and contraction in muscle. This study shows that the terminal truncation of the nNOS binding domain in the “therapeutic” del45–55 dystrophin is not innocuous since, in some cases, it may perturb the nNOS dependent stability of the RyR1/calstabin-1 complex. [Copyright &y& Elsevier]