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DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reactionor high-throughput RNA sequencingmethods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

Background and ObjectivesTo describe the phenotypic spectrum of dystrophinopathy in a large cohort of individuals with DMD exon 2 duplications (Dup2), who may be particularly amenable to therapies directed at restoring expression of either full-length dystrophin or nearly full-length dystrophin through utilization of the DMD exon 5 internal ribosome entry site (IRES).MethodsIn this retrospective observational study, we analyzed data from large genotype–phenotype databases (the United Dystrophinopathy Project [UDP] and the Italian DMD network) and classified participants into Duchenne muscular dystrophy (DMD), intermediate muscular dystrophy (IMD), or Becker muscular dystrophy (BMD) phenotypes. Log-rank tests for time-to-event variables were used to compare age at loss of ambulation (LOA) in participants with Dup2 vs controls without Dup2 in the UDP database and for comparisons between steroid-treated vs steroid-naive participants with Dup2.ResultsAmong 66 participants with Dup2 (UDP = 40, Italy = 26), 61% were classified as DMD, 9% as IMD, and 30% as BMD. Median age at last observation was 15.4 years (interquartile range 8.79–26.0) and 75% had been on corticosteroids for at least 6 months. Age at LOA differed significantly between participants with Dup2 DMD and historical controls without Dup2 DMD (p < 0.001). Valid spirometry was limited but suggested a delay in the typical age-related decline in forced vital capacity and 24 of 55 participants with adequate cardiac data had cardiomyopathy.DiscussionSome patients with Dup2 display a milder disease course than controls without Dup2 DMD, and prolonged ambulation with corticosteroids suggests the potential of IRES activation as a molecular mechanism. As Dup2-targeted therapies reach clinical applications, this information is critical to aid in the interpretation of the efficacy of new treatments.

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report o...

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial.

Background Eteplirsen, the first FDA-approved RNA-modifying therapy for DMD, is applicable to ∼13% of patients with DMD. Because multiple exonic deletions are amenable to exon 51 skipping, the isoforms resulting from the various exon 51-skipped transcripts may vary in stability, function, and phenotype. Objective/methods We conducted a detailed review of dystrophinopathy published literature and unpublished databases to compile phenotypic features of patients with exon 51 "skip-equivalent" deletions. Results Theoretically, 48 different in-frame transcripts may result from exon 51 skipping. We found sufficient clinical information on 135 patients carrying mutations that would result in production of 11 (23%) of these transcripts, suggesting the remainder have not been identified in vivo. The majority had mild phenotypes: BMD (n = 81) or isolated dilated cardiomyopathy (n = 3). Particularly interesting are the asymptomatic (n = 10) or isolated hyperCKemia (n = 20) patients with deletions of exons 45- 51, 48- 51, 49- 51 and 50- 51. Finally, 16 (12%) had more severe phenotypes described as intermediate (n = 2) or DMD (n = 14), and 6 reports had no definitive phenotype. Conclusions This review shows that the majority of exon 51 "skip-equivalent" deletions result in milder (BMD) phenotypes and supports that exon 51 skipping therapy could provide clinical benefit, although we acknowledge that other factors, such as age at treatment initiation or ongoing standard of care, may influence the degree of benefit.

The bicaudal D cargo adaptor 2 (BICD2) gene encodes a conserved cargo adaptor protein required for dynein-mediated transport. Inherited and de novo variants in BICD2 cause SMALED2 (spinal muscular atrophy lower extremity dominant 2), and a subset have recently been reported to cause severe, often lethal disease. However, a true genotype-phenotype correlation for BICD2 has not been performed, and cases described to date are scattered among at least 14 publications. In this review, we identify the characteristics of disease-causing variants in BICD2 that distinguish them from benign variation and perform genotype-phenotype correlations for 99 BICD2 variant carriers from 35 families. ANN NEUROL 2020;87:487-496.

Pontocerebellar hypoplasia type 9 (PCH9) is an autosomal recessive neurodevelopmental disorder caused by pathogenic variants in the AMPD2 gene. We evaluated the son of a consanguineous couple who presented with profound hypotonia and global developmental delay. Other features included sensorineural hearing loss, asymmetric astigmatism, and high myopia. Clinical whole-exome sequence analysis identified a homozygous missense variant in AMPD2 (NM_001257360.1:c.2201C > T, p.[Pro734Leu]) that has not been previously reported. Given the strong phenotypic overlap with PCH9, including the identification of the typical "Figure 8" appearance of the brainstem on neuroimaging, we suspect this variant was causative of the neurodevelopmental disability in this individual. An additional homozygous nonsense variant in COL11A1 (NM_001854.4:c.1168G > T, p.[Glu390Ter]) was identified. Variants in this alternatively spliced region of COL11A1 have been identified to cause an autosomal recessive form of Stickler syndrome type 2 characterized by sensorineural hearing loss and eye abnormalities, but without musculoskeletal abnormalities. The COL11A1 variant likely also contributed to the individual's phenotype, suggesting two potentially relevant genetic findings. This challenging case highlights the importance of detailed phenotypic characterization when interpreting whole exome data.

Aim: Compare efficacies of deflazacort and prednisone/prednisolone in providing clinically meaningful delays in loss of physical milestones in patients with nonsense mutation Duchenne muscular dystrophy. Materials & methods: Placebo data from Phase IIb (ClinicalTrials.gov Identifier: NCT00592553) and ACT DMD (ClinicalTrials.gov Identifier: NCT01826487) ataluren nonsense mutation Duchenne muscular dystrophy clinical trials were retrospectively combined in meta-analyses (intent-to-treat population; for change from baseline to week 48 in 6-min walk distance [6MWD] and timed function tests). Results: Significant improvements in change in 6-min walk distance with deflazacort versus prednisone/prednisolone (least-squares mean difference 39.54 m [95% CI: 13.799, 65.286; p = 0.0026]). Significant and clinically meaningful improvements in 4-stair climb and 4-stair descend for deflazacort versus prednisone/prednisolone. Conclusion: Deflazacort provides clinically meaningful delays in loss of physical milestones over 48 weeks compared with prednisone/prednisolone for patients with nonsense mutation Duchenne muscular dystrophy.

The major determinant of disease severity in patients with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD) is whether their dystrophin gene (DMD) mutation disrupts the mRNA reading frame or allows expression of a partially functional protein. However, even in the complete absence of dystrophin, variability in disease severity is observed, and candidate gene studies have implicated several genes as possible modifiers. Our previous genome-wide association study (GWAS) for age at loss of ambulation (LOA) in DMD provided confirmation for the role of genetic modifiers of TGF-β signaling in disease progression. Here we present the largest genome-wide search to date for loci influencing disease severity in DMD patients. Availability of subjects for such studies is still quite limited, leading to modest sample sizes, which present a challenge for GWAS design. We have therefore taken special steps to minimize heterogeneity within our dataset at the DMD locus itself, taking a novel and conservative approach to mutation classification to effectively exclude the possibility of residual dystrophin expression. We have also utilized statistical methods that are well adapted to smaller sample sizes, including the use of a novel linear regression-like residual for time to ambulatory loss and the application of evidential statistics for the GWAS approach. Based on the resulting sample size of N = 419 patients, we have identified multiple potential candidate genetic modifier loci. In a companion paper to this one, we use a systematic bioinformatic pipeline to implicate specific genes within these loci as potential DMD modifiers.

Although the major determinant of disease severity in patients with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD) is whether their dystrophin gene (DMD) mutation disrupts the mRNA reading frame or allows expression of a partially functional protein, other genes have been proposed or demonstrated to modify the severity of disease progression. In a companion paper to this one, we describe our novel approaches to genome-wide association study (GWAS) of loss of ambulation (LOA) in the largest genome-wide search to date for loci influencing disease severity in DMD patients. Candidate regulatory SNPs that modify disease progression were identified using an evidential statistical paradigm and here we present a uniform application of recent functional genomic datasets to explore the potential functional impact of the top six candidate regions with PPLD scores of ≥0.4. The results of this analysis of the largest DMD GWAS survey to date elucidate recurrent and potentially new pathways for intervention in the dystrophinopathies.

In a phase 1/2, open-label dose escalation trial, we delivered rAAVrh74.MCK.

Aims Dystrophin, the protein product of the DMD gene, plays a critical role in muscle integrity by stabilizing the sarcolemma during contraction and relaxation. The DMD gene is vulnerable to a variety of mutations that may cause complete loss, depletion, or truncation of the protein, leading to Duchenne and Becker muscular dystrophies. Precise and reproducible dystrophin quantification is essential in characterising DMD mutations and evaluating the outcome of efforts to induce dystrophin through gene therapies. Immunofluorescence microscopy offers high sensitivity to low levels of protein expression along with confirmation of localization, making it a critical component of quantitative dystrophin expression assays. Methods We have developed an automated and unbiased approach for precise quantification of dystrophin immunofluorescence in muscle sections. This methodology uses microscope images of whole-tissue sections stained for dystrophin and spectrin to measure dystrophin intensity and the proportion of dystrophin-positive coverage at the sarcolemma of each muscle fibre. To ensure objectivity, the thresholds for dystrophin and spectrin are derived empirically from non-sarcolemmal signal intensity within each tissue section. Furthermore, this approach is readily adaptable for measuring fibre morphology and other tissue markers. Results Our method demonstrates the sensitivity and reproducibility of this quantification approach across a wide range of dystrophin expression in both dystrophinopathy patient and healthy control samples, with high inter-operator concordance. Conclusion As efforts to restore dystrophin expression in dystrophic muscle bring new potential therapies into clinical trials, this methodology represents a valuable tool for efficient and precise analysis of dystrophin and other muscle markers that reflect treatment efficacy.

Introduction/aims In comparative studies, treatment effects are typically evaluated at a specific time point. When data are collected periodically, an alternative, clinically meaningful approach could be used to assess the totality of treatment effects. We applied a well-developed analytical procedure for evaluating longitudinal treatment effects using North Star Ambulatory Assessment (NSAA) data for illustration. Methods The NSAA comprises 17 scorable items/outcomes that measure changes in motor function. Using NSAA data from the published ataluren phase 3, randomized, placebo-controlled trial (NCT01826487), cumulative counts of failures to perform each item (transition from 2/1 [able/impaired] to 0 [unable]) were collected at specified time points for each patient over 48 wk. Treatment group-wise mean cumulative item failure count curves were constructed, comparing ataluren versus placebo and deflazacort versus prednisone/prednisolone among placebo-treated patients. The steeper the curve, the worse the outcome. A clinically meaningful summary of the between-group difference was provided for each comparison. Results The curve was uniformly steeper for placebo than ataluren after 16 wk and for prednisone/prednisolone than deflazacort after 8 wk. The two curves in each comparison continued to diverge thereafter, indicating sustained treatment benefits over time. Using a unique analytical approach, cumulative failure rates were reduced, on average, by 27% for ataluren versus placebo (rate ratio, 0.73; 95% confidence interval [CI], 0.55-0.97; p = .027) and 28% for deflazacort versus prednisone/prednisolone (rate ratio, 0.72; 95% CI, 0.53-0.96; p = .028). Discussion Unlike fixed-time analyses, this analytical approach enabled demonstration of cumulative, longitudinal treatment effects over time using repeatedly measured NSAA observations.

Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of non-coding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of DMD exon 2. We assessed the specificity of splice variation induced by AAV9.U7-ACCA delivery in the Dmd exon 2 duplication (Dup2) mouse model via an unbiased RNA-seq approach. Treatment-related effects on pre-mRNA splicing were quantified using local splicing variation (LSV) analysis. Filtering the transcriptome for differences in treatment-related splicing resulted in only 16 candidate off-target LSVs. Only a single candidate off-target LSV was found in both tissues and occurred at a known variable cassette exon. In contrast, four LSVs represented significant on-target correction of Dmd exon 2 splicing and transcriptome analysis showed correction of known dystrophin-deficient gene dysregulation. We conclude that the absence of off-target splicing induced by treatment with the U7-ACCA vector supports the continued clinical development of this approach.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by loss of dystrophin protein, encoded by the DMD gene. DMD manifests early in childhood as difficulty walking, progresses to loss of ambulation by the teens, and leads to death in early adulthood. Adeno-associated virus-vectorized gene therapies to restore dystrophin protein expression using gene replacement or antisense oligonucleotide-mediated pre-mRNA splicing modulation have emerged, making great strides in uncovering barriers to gene therapies for DMD and other genetic diseases. While this first-generation of DMD therapies are being evaluated in ongoing clinical trials, uncertainties regarding durability and therapeutic efficacy prompted the development of new experimental therapies for DMD that take advantage of somatic cell gene editing. These experimental therapies continue to advance toward clinic trials, but questions remain unanswered regarding safety and translatable efficacy. Here we review the advancements toward treatment of DMD using gene editing and modulation therapies, with an emphasis on those nearest to clinical applications.

Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of noncoding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of

The purpose of this review is to highlight updates in the standard of care recommendations for DMD, and to describe approaches to and recent advances in genetic therapies for DMD.Treatment of DMD patients with the corticosteroids prednisone or deflazacort remains the standard of care, and recent data shows that early treatment (as young as 5 months) with a weekend dosing regimen results in measurable improvement in motor outcomes. A mutation-specific therapy directed at restoring an open reading frame by skipping exon 51 is FDA-approved, and therapies directed at other exons are in trials. Gene replacement therapy shows significant promise in animal models, and trials are underway. Genome editing has received significant attention because of results in animal models, but challenges to implementation in humans remain.The mainstay of treatment remains meeting well defined standards of care that have been shown to influence morbidity and mortality. These include use of systemic steroids, early nocturnal ventilatory support, appropriate cardiac care and prophylaxis, and wherever appropriate, scoliosis surgery. Early and accurate molecular diagnosis, along with appropriate and multidisciplinary care, provides the best opportunity for maximum benefit of both current standard and upcoming novel therapies for boys with DMD. Among the most promising of these is AAV-based gene replacement therapy, which is currently in clinical trials.

Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) is a childhood metabolic disorder marked by neuropathology arising due to impaired heparan sulphate (HS) catabolism. Consequently, partially degraded HS accumulates in the lysosomes of affected cells and is excreted in the urine. The measurement of HS in urine has long been considered a biomarker of Sanfilippo syndrome although it is largely non-specific. Using blood, urine and CSF collected from a cohort of Sanfilippo patients we investigated the utility of primary and secondary biomarkers to inform on disease activity. These included enzyme activity, specific oligosaccharides with non-reducing end residues reflective of the enzyme deficiency, and gangliosides. The diagnostic oligosaccharides - a HS disaccharide and tetrasaccharide - were elevated in the urine, plasma and CSF of all MPS IIIA and IIIB patients, respectively. There was no correlation between the concentrations in any of the matrices suggesting they reflect specific tissues and not overall disease burden. Enzyme activity did not inform on disease severity, with no measurable activity in CSF and activity approaching normal in MPS IIIA plasma. The concentration of gangliosides, GM2 and GM3, were significantly higher in the CSF of all MPS III subjects when compared to controls and correlated with the age of onset of first symptoms. Given that these gangliosides reflect delayed brain development they may be useful measures of disease burden, within the limitations of the clinical surrogates. Observation of these biochemical measurements in MPS III patients enrolled in clinical trials may determine whether they represent true pharmacodynamics biomarkers.

Approved treatments in spinal muscular atrophy (SMA) have resulted in unprecedented gains for many individuals. Use of available outcomes, typically developed for a specific type of SMA, do not cover the range of progression, often resulting in a battery of functional testing being completed at visits. Our objective was to validate the Neuromuscular Gross Motor Outcome (GRO) as a tool to quantify function in SMA across the span of abilities.Patients with genetically confirmed SMA completed functional testing at each visit including the Neuromuscular GRO and other appropriate gross motor outcomes.We enrolled 91 patients with SMA types 1 to 3 between 8 days and 32.1 years. The GRO utilizes a 0- to 2-point scale with scores in our cohort ranging from 1 to 95 points with no floor or ceiling effect. GRO scores were significantly different across functional categories (P 0.001) and treatment status (P = 0.01) and correlated to other functional assessments (P ≤ 0.001). All patients were measured using the GRO, whereas traditional outcomes were only appropriate on 36% to 59% of our cohort.The Neuromuscular GRO quantifies function across the span of age and abilities included in our cohort, allowing for continuous longitudinal monitoring on one scale to reduce the burden of testing in our heterogeneous clinic population.

Objective To quantify changes in segmented brain volumes over 12 months in children with mucopolysaccharidosis types IIIA and IIIB (MPS IIIA and IIIB). Methods In order to establish suitable outcome measures for clinical trials, twenty-five children greater than 2 years of age were enrolled in a prospective natural history study of MPS IIIA and IIIB at Nationwide Children's Hospital. Data from sedated non-contrast brain 3 T MRIs and neuropsychological measures were reviewed from the baseline visit and at 12-month follow-up. No intervention beyond standard clinical care was provided. Age- and sex-matched controls were gathered from the National Institute of Mental Health Data Archive. Automated brain volume segmentation with longitudinal processing was performed using FreeSurfer. Results Of the 25 subjects enrolled with MPS III, 17 children (4 females, 13 males) completed at least one MRI with interpretable volumetric data. The ages ranged from 2.8 to 13.7 years old (average 7.2 years old) at enrollment, including 8 with MPS IIIA and 9 with MPS IIIB. At baseline, individuals with MPS III demonstrated reduced cerebral white matter and corpus callosum volumes, but greater volumes of the lateral ventricles, cerebellar cortex, and cerebellar white matter compared to controls. Among the 13 individuals with MPS III with two interpretable MRIs, there were annualized losses or plateaus in supratentorial brain tissue volumes (cerebral cortex −42.10 ± 18.52 cm3/year [mean ± SD], cerebral white matter −4.37 ± 11.82 cm3/year, subcortical gray matter −6.54 ± 3.63 cm3/year, corpus callosum −0.18 ± 0.62 cm3/yr) and in cerebellar cortex (−0.49 ± 12.57 cm3/year), with a compensatory increase in lateral ventricular volume (7.17 ± 6.79 cm3/year). Reductions in the cerebral cortex and subcortical gray matter were more striking in individuals younger than 8 years of age. Greater cerebral cortex volume was associated with higher fine and gross motor functioning on the Mullen Scales of Early Learning, while greater subcortical gray matter volume was associated with higher nonverbal functioning on the Leiter International Performance Scale. Larger cerebellar cortex was associated with higher receptive language performance on the Mullen, but greater cerebellar white matter correlated with worse adaptive functioning on the Vineland Adaptive Behavioral Scales and visual problem-solving on the Mullen. Conclusions Loss or plateauing of supratentorial brain tissue volumes may serve as longitudinal biomarkers of MPS III age-related disease progression compared to age-related growth in typically developing controls. Abnormally increased cerebellar white matter in MPS III, and its association with worse performance on neuropsychological measures, suggest the possibility of pathophysiological mechanisms distinct from neurodegeneration-associated atrophy that warrant further investigation.

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an adeno-associated virus (AAV)-encapsidated viral vector containing four copies of the noncoding U7 small nuclear RNA (U7snRNA), each targeted to either the splice donor or the splice acceptor sites of

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by loss of dystrophin protein, encoded by the DMD gene. DMD manifests early in childhood as difficulty walking, progresses to loss of ambulation by the teens, and leads to death in early adulthood. Adeno-associated virus-vectorized gene therapies to restore dystrophin protein expression using gene replacement or antisense oligonucleotide-mediated pre-mRNA splicing modulation have emerged, making great strides in uncovering barriers to gene therapies for DMD and other genetic diseases. While this first-generation of DMD therapies are being evaluated in ongoing clinical trials, uncertainties regarding durability and therapeutic efficacy prompted the development of new experimental therapies for DMD that take advantage of somatic cell gene editing. These experimental therapies continue to advance toward clinic trials, but questions remain unanswered regarding safety and translatable efficacy. Here we review the advancements toward treatment of DMD using gene editing and modulation therapies, with an emphasis on those nearest to clinical applications.

Knowledge of health related quality of life (HRQOL) in the immediate phase following DMD diagnosis has not been well-characterized. It is important to understand HRQOL early in disease for both clinical care and studies of treatment. The relationship between parent-proxy and child self-report HRQOL and their associations with medical, psycho-social and behavioral symptoms deserve study. In this study HRQOL was measured using the PedsQL inventory in parent/caregiver and corticosteroid-naïve boys (ages 4 to 7 years) participating in the FOR-DMD study. Agreement between the parent-proxy report and the boys' self-report HRQOL was measured using intraclass correlation coefficients (ICCs). Factors associated with HRQOL, including standardized psychosocial and behavioral measures in this cross-sectional sample, were explored using correlations. The results showed that the level of agreement between 70 dyads of child self-report and parent-proxy ratings of HRQOL was poor for the generic PedsQL total score (ICC=0.48, 95% CI (0.23, 0.66)) and its subscale scores, and was similarly low for the neuromuscular disease module (ICC=0.24, 95% CI (0.00, 0.45)). Parents rated their child's HRQOL as poorer than the children rated themselves in all scales. Psychosocial outcome measures were more highly associated with HRQOL measures than disease severity or patient demographic variables. In the early phases of DMD, child and parent-proxy HRQOL ratings were discordant. In early DMD, psychosocial and behavioral aspects appear to be more relevant to HRQOL than disease severity factors.

Aim: Assess the totality of efficacy evidence for ataluren in patients with nonsense mutation Duchenne muscular dystrophy (nmDMD). Materials & methods: Data from the two completed randomized controlled trials (ClinicalTrials.gov: NCT00592553; NCT01826487) of ataluren in nmDMD were combined to examine the intent-to-treat (ITT) populations and two patient subgroups (baseline 6-min walk distance [6MWD] ≥300–Results: Statistically significant differences in 6MWD change with ataluren versus placebo were observed across all three meta-analyses. Least-squares mean difference (95% CI): ITT (n = 342), +17.2 (0.2–34.1) m, p = 0.0473; ≥300–Conclusion: These meta-analyses support previous evidence for ataluren in slowing disease progression versus placebo in patients with nmDMD over 48 weeks. Treatment benefit was most evident in patients with a baseline 6MWD ≥300–

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial., Graphical abstract, Flanigan and colleagues report the results of an intravenous dose-escalation study using an AAV9-encapsidated U7snRNA vector to skip DMD exon 2 and restore dystrophin expression in the Dup2 mouse model of Duchenne muscular dystrophy. The results informed dose considerations for an ongoing clinical trial.

Clinical trials targeting younger cohorts of boys with Duchenne muscular dystrophy are necessary as earlier intervention may maximize treatment effect. Boys with Duchenne muscular dystrophy often have gross motor delays very early in life, and although they gain skills, they are on a lower trajectory than typical peers. Quantifying the natural rate of motor maturation in Duchenne muscular dystrophy from an early age permits identification of deviations from the expected trajectory related to treatment effects.The purpose of our study was to define the natural history in boys aged from ≥3 to8 years using the North Star Ambulatory Assessment (NSAA), 100-meter timed test (100m), 10-meter walk/run (10m), time to rise (Rise), and 4-stair climb (4SC). Assessments were completed as standard of care during regularly scheduled clinic visits.One hundred sixty-two boys with DMD aged 3.1 to 7.9 years on glucocorticoids were evaluated using one or more of the following tests as appropriate for age: NSAA (N = 158; 3.1-7.9 years), 100m (N = 131; 3.4-7.9 years), 10m (N = 162; 3.1-7.9 years), Rise (N = 160; 3.1-7.9 years), and 4SC (N = 153; 3.1-7.9 years). Longitudinal data are presented by age in a subcohort (N = 64).Our study documents the baseline function of boys with DMD who are being treated with corticosteroids. These data will be useful to compare ongoing and future therapeutic intervention(s) for DMD.

Background Canine models of Duchenne muscular dystrophy (DMD) are valuable to evaluate therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the GRMD (Golden Retriever Muscular Dystrophy) model remains the one used in most preclinical studies. Methods We report a new spontaneous dystrophinopathy in a Labrador retriever strain, named LRMD (Labrador Retriever Muscular Dystrophy), for which a colony was established. Fourteen LRMD dogs were followed-up and compared to the GRMD standard. Results The clinical features of the GRMD disease were found in LRMD dogs, and the functional tests provided data roughly overlapping those measured in GRMD dogs, with similar inter-individual heterogeneity. Molecular techniques including RNA-sequencing allowed to map and identify the LRMD causal mutation, consisting in a 2.2-Mb inversion disrupting the DMD gene within its intron 20, and involving TMEM47 gene. In skeletal muscles, the Dp71 isoform was ectopically expressed as a probable consequence of the mutation. We found no evidence of polymorphism in the two LTBP4 and Jagged1 modifier genes that would explain the observed inter-individual variability. Conclusions This study provides a full comparative description of a new spontaneous canine dystrophinopathy, that we demonstrate is phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes.

Background Canine models of Duchenne muscular dystrophy (DMD) are a valuable tool to evaluate potential therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the Golden Retriever muscular dystrophy (GRMD) model remains the most used in preclinical studies. Here, we report a new spontaneous dystrophinopathy in a Labrador Retriever strain, named Labrador Retriever muscular dystrophy (LRMD). Methods A colony of LRMD dogs was established from spontaneous cases. Fourteen LRMD dogs were followed-up and compared to the GRMD standard using several functional tests. The disease causing mutation was studied by several molecular techniques and identified using RNA-sequencing. Results The main clinical features of the GRMD disease were found in LRMD dogs; the functional tests provided data roughly overlapping with those measured in GRMD dogs, with similar inter-individual heterogeneity. The LRMD causal mutation was shown to be a 2.2-Mb inversion disrupting the DMD gene within intron 20 and involving the TMEM47 gene. In skeletal muscle, the Dp71 isoform was ectopically expressed, probably as a consequence of the mutation. We found no evidence of polymorphism in either of the two described modifier genes LTBP4 and Jagged1. No differences were found in Pitpna mRNA expression levels that would explain the inter-individual variability. Conclusions This study provides a full comparative description of a new spontaneous canine model of dystrophinopathy, found to be phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes.

Recombinant adeno-associated virus (rAAV)rh74.MCK.GALGT2 is a muscle-specific gene therapy that is being developed to treat forms of muscular dystrophy. Here we report on an isolated limb infusion technique in a non-human primate model, where hindlimb blood flow is transiently isolated using balloon catheters to concentrate vector in targeted leg muscles. A bilateral dose of 2.5 × 1013 vector genomes (vg)/kg/limb was sufficient to induce GALGT2-induced glycosylation in 10%–60% of skeletal myofibers in all leg muscles examined. There was a 19-fold ± 6-fold average limb-wide increase in vector genomes per microgram genomic DNA at a bilateral dose of 2.5 × 1013 vg/kg/limb compared with a bilateral dose of 6 × 1012 vg/kg/limb. A unilateral dose of 6 × 1013 vg/kg/limb showed a 12- ± 3-fold increase in treated limb muscles compared to contralateral untreated limb muscles, which received vector only after release into the systemic circulation from the treated limb. Variability in AAV biodistribution between different segments of the same muscle was 125% ± 18% for any given dose, while variability between the same muscle for any given treatment dose was 45% ± 7%. These experiments demonstrate that treatment of muscles throughout the leg with rAAVrh74.MCK.GALGT2 can be accomplished safely using an isolated limb infusion technique, where balloon catheters transiently isolate the limb vasculature, but that intra- and inter-muscle transduction variability is a significant issue. Keywords: limb perfusion, gene therapy, Galgt2, muscular dystrophy

OBJECTIVE Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by loss-of-function dystrophin (DMD) mutations in boys, who typically suffer loss of ambulation by age 12. Previously, we reported that coding variants in latent transforming growth factor beta (TGFβ)-binding protein 4 (LTBP4) were associated with reduced TGFβ signaling and prolonged ambulation (p = 1.0 × 10-3 ) in DMD patients; this result was subsequently replicated by other groups. In this study, we evaluated whether additional DMD modifier genes are observed using whole-genome association in the original cohort. METHODS We performed a genome-wide association study (GWAS) for single-nucleotide polymorphisms (SNPs) influencing loss of ambulation (LOA) in the same cohort of 253 DMD patients used to detect the candidate association with LTBP4 coding variants. Gene expression and chromatin interaction databases were used to fine-map association signals above the threshold for genome-wide significance. RESULTS Despite the small sample size, two loci associated with prolonged ambulation met genome-wide significance and were tagged by rs2725797 (chr15, p = 6.6 × 10-9 ) and rs710160 (chr19, p = 4.7 × 10-8 ). Gene expression and chromatin interaction data indicated that the latter SNP tags regulatory variants of LTBP4, whereas the former SNP tags regulatory variants of thrombospondin-1 (THBS1): an activator of TGFβ signaling by direct binding to LTBP4 and an inhibitor of proangiogenic nitric oxide signaling. INTERPRETATION Together with previous evidence implicating LTBP4, the THBS1 modifier locus emphasizes the role that common regulatory variants in gene interaction networks can play in mitigating disease progression in muscular dystrophy. Ann Neurol 2018;84:234-245.

Objective This double‐blind, randomized, placebo‐controlled Phase 2 study (NCT01462292) assessed the 24‐week efficacy, safety, tolerability, and pharmacokinetics of two different subcutaneous drisapersen doses, and the 24‐week off‐dose persistent effect, in ambulant Duchenne muscular dystrophy (DMD) patients. Methods Male DMD patients (≥5 years; time to rise from floor ≤15 s) were randomized to drisapersen 3 mg/kg/week, 6 mg/kg/week or placebo. The primary efficacy endpoint was change from baseline in 6‐minute walking distance (6MWD) at week 24. Secondary endpoints included changes in timed function tests, muscle strength, and pulmonary function tests. Results Fifty‐one patients were randomized to placebo (N = 16), drisapersen 3 mg/kg/week (N = 17) or 6 mg/kg/week (N = 18). All but 2 patients had baseline rise from floor time

The reading frame rule suggests that Duchenne muscular dystrophy (DMD) results from DMD mutations causing an out-of-frame transcript, whereas the milder Becker muscular dystrophy results from mutations causing an in-frame transcript. However, predicted nonsense mutations may instead result in altered splicing and an in-frame transcript. Here we report a 10-year-old boy with a predicted nonsense mutation in exon 42 who had a 6-minute walk time of 157% of that of age matched DMD controls, characterized as intermediate muscular dystrophy. RNA sequencing analysis from a muscle biopsy revealed only 6.0–9.8% of DMD transcripts were in-frame, excluding exon 42, and immunoblot demonstrated only 3.2% dystrophin protein expression. Another potential genetic modifier noted was homozygosity for the protective IAAM LTBP4 haplotype. This case suggests that very low levels of DMD exon skipping and dystrophin protein expression may result in amelioration of skeletal muscle weakness, a finding relevant to current dystrophin-restoring therapies.