Duchenne Muscular Dystrophy Genotypes Research Articles

Appendicular lean mass index changes in patients with Duchenne muscular dystrophy and Becker muscular dystrophy.

Mutations in the 79 exons of the dystrophin gene result in muscle wasting and weakness of varying clinical severity, ranging from severe/typical Duchenne muscular dystrophy (DMD) to intermediate DMD and mild Becker muscular dystrophy (BMD), depending on the frameshift of the mutation. We previously reported that males with DMD have progressively declining appendicular lean mass (ALM) and ALM index (ALMI) with age and worsening functional motor ability compared with healthy controls. These indices have not been studied in patients with intermediate DMD and BMD phenotypes and across DMD genotypes. In this study, we compared age-related trajectories of ALM and ALMI of patients who had (1) BMD without functional mobility deficits with patients who had DMD at different stages of disease and healthy controls; (2) a DMD intermediate phenotype with patients who had a typical DMD phenotype; and (3) DMD categorized by genotype. We conducted a retrospective review of ALM and ALMI data from 499 patients (ages 5-23years) with DMD (466 typical and 33 intermediate) and 46 patients (ages 5-21years) with BMD (without functional mobility deficits and functional mobility score of 1). Patients were grouped according to age reflecting disease stage (ages 5 to <7, 7 to <10, 10 to <14, and 14 to <20years) and genotype (mutations in exons 1-30, 31-44, 45-62, and 63-79). ALM and ALMI trajectories of patients with BMD paralleled those of healthy controls until adolescence, in contrast to patients with DMD. ALMI Z-scores of patients with BMD remained within ±2 SD without decline while those of patients with DMD fell below -2 SD around age 12years. Patients with BMD had increasing ALM and ALMI with age, with peak accrual between ages 10 to <14years. ALMI declined after age 14years for those with intermediate DMD compared with 10years for patients with typical DMD. Patients with mutations in exons 63-79 had a greater decline in ALMI as compared with those with other genotypes after age 10years. Age-related changes in ALMI in patients with BMD and intermediate DMD differ from those with typical DMD, reflecting their clinical phenotypes. ALM and ALMI should be further studied in patients with BMD and DMD subtypes for their potential value as surrogate markers to characterize the severity of BMD and DMD and inform clinical care decisions and clinical trial designs.

DMD Genotypes and Motor Function in Duchenne Muscular Dystrophy: A Multi-institution Meta-analysis With Implications for Clinical Trials.

Clinical trials of genotype-targeted treatments in Duchenne muscular dystrophy (DMD) traditionally compare treated patients with untreated patients with the same DMD genotype class. This avoids confounding of drug efficacy by genotype effects but also shrinks the pool of eligible controls, increasing challenges for trial enrollment in this already rare disease. To evaluate the suitability of genotypically unmatched controls in DMD, we quantified effects of genotype class on 1-year changes in motor function endpoints used in clinical trials. More than 1,600 patient-years of follow-up (>700 patients) were studied from 6 real-world/natural history data sources (UZ Leuven, PRO-DMD-01 shared by CureDuchenne, iMDEX, North Star UK, Cincinnati Children's Hospital Medical Center, and the DMD Italian Group), with genotypes classified as amenable to skipping exons 44, 45, 51, or 53, or other skippable, nonsense, and other mutations. Associations between genotype class and 1-year changes in North Star Ambulatory Assessment total score (ΔNSAA) and in 10-m walk/run velocity (Δ10MWR) were studied in each data source with and without adjustment for baseline prognostic factors. The studied genotype classes accounted for approximately 2% of variation in ΔNSAA outcomes after 12 months, whereas other prognostic factors explained >30% of variation in large data sources. Based on a meta-analysis across all data sources, pooled effect estimates for the studied skip-amenable mutation classes were all small in magnitude (<2 units in ΔNSAA total score in 1-year follow up), smaller than clinically important differences in NSAA, and were precisely estimated with standard errors <1 unit after adjusting for nongenotypic prognostic factors. These findings suggest the viability of trial designs incorporating genotypically mixed or unmatched controls for up to 12 months in duration for motor function outcomes, which would ease recruitment challenges and reduce numbers of patients assigned to placebos. Such trial designs, including multigenotype platform trials and hybrid designs, should ensure baseline balance between treatment and control groups for the most important prognostic factors, while accounting for small remaining genotype effects quantified in this study.

The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45-47.

Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy(DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P<0.0001), wire hang (P=0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P=0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P<0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P=0.0017) and centrally located nuclei indicating degeneration/regeneration (P<0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P<0.0001), collagen deposition (+1.4-fold, P=0.0217), and sarcolemmal damage measured by intracellular IgM (P=0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

P.126 Genotype-unmatched controls are feasible for drug development in Duchenne muscular dystrophy (DMD)

Clinical trials of genotype-targeted therapies in DMD typically compare treated patients to genotype-matched controls. While this avoids confounding by genotype effects, the pool of eligible controls is further limited in an already rare disease. We hypothesized that using genotype-unmatched controls can be feasible when 1) DMD genotype class effects are small and precisely known and 2) stronger baseline prognostic factors are accounted for. We evaluated this prospect in a hypothetical clinical trial for effects on 1-year change in the North Star Ambulatory Assessment total score (ΔNSAA). The hypothetical ‘treatment' group (n=58) was exon 51 skip-amenable patients from the DEMAND III trial placebo arm. The genotype-unmatched ‘control' group (n=58) included patients amenable to skipping exons other than 44, 45, 51 or 53 drawn from five natural history studies using similar age, function, and steroid inclusion criteria. Mean difference in ΔNSAA for the treatment vs. control group indicated a bias of -1.6 NSAA units. After statistically accounting for genotype differences and baseline prognostic factors (e.g., motor function and steroid type), the bias was reduced to -0.3 units. As a statistical cost of genotype adjustment, the standard error of the treatment effect increased by 35%. Power analysis indicated that detecting a 5 (or 4) unit treatment effect using unmatched vs. matched controls would require 1.2 (or 1.4) times more enrolled patients. When genotype adjustment was integrated into a hypothetical multi-genotype, randomized, double-blind platform trial, overall sample size requirements were reduced vs. conducting separate trials with matched controls for each genotype class. We conclude that comparisons with genotypically-unmatched controls, with careful consideration of baseline prognostic factors, are feasible in DMD drug development and could help limit placebo exposure among patients eligible for genotype-targeted treatments.

Genotype-related respiratory progression in Duchenne muscular dystrophy-A multicenter international study.

Mutations amenable to skipping of specific exons have been associated with different motor progression in Duchenne muscular dystrophy (DMD). Less is known about their association with long-term respiratory function. In this study we investigated the features of respiratory progression in four DMD genotypes relevant in ongoing exon-skipping therapeutic strategies. This was a retrospective longitudinal study including DMD children followed by the UK NorthStar Network and international AFM Network centers (May 2003 to October 2020). We included boys amenable to skip exons 44, 45, 51, or 53, who were older than 5 years of age and ambulant at first recorded visit. Subjects who were corticosteroid-naive or enrolled in interventional clinical trials were excluded. The progression of respiratory function (absolute forced vital capacity [FVC] and calculated as percent predicted [FVC%]) was compared across the four subgroups (skip44, skip45, skip51, skip53). We included 142 boys in the study. Mean (standard deviation) age at first visit was 8.6 (2.5) years. Median follow-up was 3 (range, 0.3-8.3) years. In skip45 and skip51, FVC% declined linearly from the first recorded visit. From the age of 9 years, FVC% declined linearly in all genotypes. Skip44 had the slowest (2.7%/year) and skip51 the fastest (5.9%/year) annual FVC% decline. The absolute FVC increased progressively in skip44, skip45, and skip51. In skip53, FVC started declining from 14 years of age. The progression of respiratory dysfunction follows different patterns for specific genotype categories. This information is valuable for prognosis and for the evaluation of exon-skipping therapies.

Genetic Modifiers of Duchenne Muscular Dystrophy in Chinese Patients.

Background: Duchenne muscular dystrophy (DMD) is a fatal, X-linked recessive muscle disorder characterized by heterogeneous progression and severity. We aimed to study the effects of single nucleotide polymorphisms (SNPs) in SPP1 and LTBP4 on DMD progression in Chinese patients.Methods: We genotyped LTBP4 haplotypes and the SPP1 promoter SNPs rs28357094, rs11730582, and rs17524488 in 326 patients registered in the neuromuscular database of The First Affiliated Hospital of Sun Yat-sen University. Kaplan-Meier curves and log-rank tests were used to estimate and compare median age at loss of ambulation, while Cox proportional hazard regression models were used as to analyze the effects of glucocorticoids treatments, DMD genotype, and SPP1/LTBP4 SNPs on loss of ambulation.Results: The CC/CT genotype at rs11730582 was associated with a 1.33-year delay in ambulation loss (p = 0.006), with hazard ratio 0.63 (p = 0.008), in patients with truncated DMD genotype and undergoing steroid treatment. On the other hand, rs17524488 in SPP1 and the IAAM/IAAM haplotype in LTBP4 were not associated with time to ambulation loss.Conclusions: SPP1 rs11730582 is a genetic modifier of the long-term effects of steroid treatment in Chinese DMD patients. Thus, any future clinical study in DMD should adjust for glucocorticoids use, DMD genotype, and SPP1 polymorphisms.

PRO65 VARIABILITY IN AGE AT LOSS OF AMBULATION BY GENOTYPE AMONG BOYS WITH DUCHENNE MUSCULAR DYSTROPHY

Duchenne muscular dystrophy (DMD), caused by loss of dystrophin, is associated with early loss of ambulation (LOA) and death. While severities of clinical phenotypes are correlated with underlying genotypes, a full synthesis of these relationships is lacking. This review’s objective was to summarize published data for age at LOA with DMD genotype. A systematic review using MEDLINE and EMBASE identified articles describing the natural history among patients with known DMD genotype. To focus on a more homogeneous group, 1) this analysis was restricted to patients treated with corticosteroids (CS) and 2) DMD genotypes were categorized according to amenability of mutations to potential exon skipping therapies. Outcomes included mean (95% confidence interval [CI]) and median age at LOA, as defined by original investigators. From 7,291 publications, 8 studies met inclusion criteria. Five studies presented individual patient data on age at LOA. Mean (95%CI) age at LOA ranged from 10.1 (9.9-10.2; n=2, exon 52 skip amenable) and 10.2 (9.4-11.1; n=16, exon 53 skip amenable) years; to 12.6 (10.8-14.3) years (n=13, exon 44 skip amenable). Median age at LOA ranged from 10.0 (n=3, exon 53 skip amenable) to 12.0 (n=2, exon 55 skip amenable) years. Three studies reported median age at LOA from samples where not everyone had experienced LOA; estimates ranged from 12.0 (n=27, exon 45 skip amenable) to 20 (n=74, exon 44 skip amenable) years. Variability in LOA age by DMD genotype was observed. Those amenable to exon 52 and 53 skipping showed more severe phenotypes in terms of age at LOA compared to those with exon 55 or 44 skip amenable mutations. These findings are consistent with published estimates from large registries. Caution is needed when comparing LOA from studies where designs and inclusion criteria differed. Furthermore, the natural history of certain genotypes is better characterized.

Haplotype-Based noninvasive prenatal diagnosis for duchenne muscular dystrophy: A pilot study in South China

Objective: To explore the accuracy and feasibility of noninvasive prenatal diagnosis (NIPD) for Duchenne Muscular Dystrophy (DMD) based on the haplotype approach.Methods: We recruited singleton pregnancies at-risk of DMD at 12–25 weeks of gestation from 17 families who all had a proband child affected by DMD. We have identified the pathogenic mutations in probands and their mothers by multiplex ligation-dependent probe amplification (MLPA). To construct parental haplotypes, we performed captured sequencing on genomic DNA from parents and probands. The integration analysis of parental haplotypes and targeted sequencing results of maternal plasma DNA were used to infer the fetal haplotype and genotypes in the DMD gene. Fetal DMD genotypes were further confirmed by invasive prenatal diagnosis.Result: We have successfully performed the haplotype-based NIPD in all recruited families. Ten fetuses were identified as normal, including four female and six male fetuses. Four female fetuses were carriers, and the other three male fetuses were affected by DMD with exons 49–52 deletion, exons 8–37 deletion and c.628 G > T mutation, respectively. The results of NIPD were consistent with those of invasive diagnosis.Conclusion: Haplotype-based NIPD for DMD by targeted sequencing is promising and has the potential for clinical application.

DMD genotype correlations from the Duchenne Registry: Endogenous exon skipping is a factor in prolonged ambulation for individuals with a defined mutation subtype.

Antisense oligonucleotide (AON)‐mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in‐frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3–7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3–7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.

DMD mutation and LTBP4 haplotype do not predict onset of left ventricular dysfunction in Duchenne muscular dystrophy.

Cardiomyopathy develops in >90% of Duchenne muscular dystrophy (DMD) patients by the second decade of life. We assessed the associations between DMD gene mutations, as well as Latent transforming growth factor-beta-binding protein 4 (LTBP4) haplotypes, and age at onset of myocardial dysfunction in DMD. DMD patients with baseline normal left ventricular systolic function and genotyping between 2004 and 2013 were included. Patients were grouped in multiple ways: specific DMD mutation domains, true loss-of-function mutations (group A) versus possible residual gene expression (group B), and LTBP4 haplotype. Age at onset of myocardial dysfunction was the first echocardiogram with an ejection fraction <55% and/or shortening fraction <28%. Of 101 DMD patients, 40 developed cardiomyopathy. There was no difference in age at onset of myocardial dysfunction among DMD genotype mutation domains (13.7±4.8 versus 14.3±1.0 versus 14.3±2.9 versus 13.8±2.5, p=0.97), groups A and B (14.4±2.8 versus 12.1±4.4, p=0.09), or LTBP4 haplotypes (14.5±3.2 versus 13.1±3.2 versus 11.0±2.8, p=0.18). DMD gene mutations involving the hinge 3 region, actin-binding domain, and exons 45-49, as well as the LTBP4 IAAM haplotype, were not associated with age of left ventricular dysfunction onset in DMD.

Natural history of a cohort of Duchenne muscular dystrophy children seen between 1998 and 2014: An observational study from South India.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. There are no large studies describing its natural course from India. Immunohistochemically/genetically confirmed DMD patients diagnosed between 1998 and 2014 were ambispectively included. The main aim was to study the natural course of motor milestones, i.e., age at onset of wheelchair status, bedbound state, and age at death, which were considered as primary outcome measures. We also correlated the DMD genotype with the motor milestones and other phenotypic features. A total of 500 DMD patients were included and 275 participated in the study. The mean age at symptom onset was 3.7 ± 1.9 years, mean age at presentation was 8.1 ± 2.5 years, and mean duration of illness was 4.4 ± 2.6 years. On following them over 15 years, 155 (56.4%) had attained at least one of the primary outcome measures. Wheelchair status was attained in 124 (45.1%) [mean age: 10.4 ± 1.6 years] and bedbound state in 24 (8.7%; mean age: 11.8 ± 2.2 years) patients. Seven patients (2.6%) died during the follow-up period (mean age: 15.2 ± 2.4 years). There was no significant impact of the genotypic or phenotypic features on the primary outcome. The pattern of major motor milestones (primary outcome measures) in this large cohort is comparable with that of the Western population despite variability in medical care. The genotypic pattern was also similar to other large studies, which suggests that DMD is a more homogeneous disorder with limited ethnic variability in its geno-phenotypic expression.

Assessing mental health in boys with Duchenne muscular dystrophy: Emotional, behavioural and neurodevelopmental profile in an Italian clinical sample.

To evaluate through a comprehensive protocol, the psychopathological profile of DMD boys. The primary aim of this observational study was to describe the emotional and behavioural profile and the neurodevelopmental problems of Italian boys with Duchenne Muscular Dystrophy (DMD); the secondary aim was to explore the relation between psychopathological profile and DMD genotype. 47 DMD boys, aged 2-18, were included in the study and assessed through structured and validated tools including Wechsler scales or Griffiths for cognitive ability, Child Behavior Check List (CBCL), Youth Self Report (YSR) and Strengths and Difficulties Questionnaire (SDQ) for emotional and behavioural features. Patients "at risk" based on questionnaires scores were evaluated by a clinical structured interview using Development and Well Being Assessment (DAWBA) or Autism Diagnostic Observation Schedule (ADOS), as required. The 47 enrolled patients, defined with a Full Scale Intelligence Quotient (FSIQ) of 80.38 (one SD below average), and presenting a large and significant difference in FSIQ in relation to the site of mutation along the dystrophin gene (distal mutations associated with a more severe cognitive deficit), were showing Internalizing Problems (23.4%) and Autism Spectrum Disorders (14.8%). Interestingly, an association of internalizing problems with distal deletion of the DMD gene is documented. Even though preliminary, these data show that the use of validated clinical instruments, that focus on the impact of emotional/behaviour problems on everyday life, allows to carefully identify clinically significant psychopathology.

P.103 - DMD genotypes and loss of ambulation in the CINRG Duchenne natural history study

Objective: To correlate time to loss of ambulation (LoA) and different truncating DMD gene mutations in a large, prospective natural history study of Duchenne muscular dystrophy (DMD), with particular attention to mutations amenable to emerging molecular treatments. Methods: We analyzed data from the Cooperative International Neuromuscular Research Group Duchenne Natural History Study for participants with DMD single- or multi-exon deletions or duplications with defined exon boundaries (n = 186), or small mutations identified by sequencing (n = 26, including 16 nonsense point mutations). We performed a time-to-event analysis of LoA, a strong indicator of overall disease severity, adjusting for glucocorticoid treatment and genetic modifiers. Results: Participants with deletions amenable to skipping of exon 44 had later LoA (median 14.8 years, hazard ratio 0.31, 95% confidence interval 0.14–0.69, p = 0.004). Age at LoA did not differ significantly in participants with deletions amenable to exon 45, 51, and 53 skipping, duplications, and small rearrangements. Nonsense mutation DMD also showed a typical median age at LoA (11.1 years), with a few outliers (ambulatory around or after 16 years of age) carrying stop codons within in-frame exons, more often situated in the rod domain. Conclusions: As exon 44 skipping–amenable DMD has a later LoA, mutation-specific randomization and selection of placebo groups are essential for the success of clinical trials.

DMD genotypes and loss of ambulation in the CINRG Duchenne Natural History Study.

To correlate time to loss of ambulation (LoA) and different truncating DMD gene mutations in a large, prospective natural history study of Duchenne muscular dystrophy (DMD), with particular attention to mutations amenable to emerging molecular treatments. We analyzed data from the Cooperative International Neuromuscular Research Group Duchenne Natural History Study for participants with DMD single- or multi-exon deletions or duplications with defined exon boundaries (n = 186), or small mutations identified by sequencing (n = 26, including 16 nonsense point mutations). We performed a time-to-event analysis of LoA, a strong indicator of overall disease severity, adjusting for glucocorticoid treatment and genetic modifiers. Participants with deletions amenable to skipping of exon 44 had later LoA (median 14.8 years, hazard ratio 0.31, 95% confidence interval 0.14-0.69, p = 0.004). Age at LoA did not differ significantly in participants with deletions amenable to exon 45, 51, and 53 skipping, duplications, and small rearrangements. Nonsense mutation DMD also showed a typical median age at LoA (11.1 years), with a few outliers (ambulatory around or after 16 years of age) carrying stop codons within in-frame exons, more often situated in the rod domain. As exon 44 skipping-amenable DMD has a later LoA, mutation-specific randomization and selection of placebo groups are essential for the success of clinical trials.

317. Screening S. Aureus CRISPR-Cas9 Paired-Guide RNAs for Efficient Targeted Deletion in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a recessive X-linked neuromuscular disorder that results in progressive muscle degeneration and premature death. Most patients have exonic deletions in the dystrophin gene that result in a frameshift and nonfunctional protein. In contrast, Becker muscular dystrophy (BMD) patients carry a range of exonic deletions in dystrophin that do not disrupt the reading frame, leading to a much milder disease phenotype. Thus, multiplex CRISPR/Cas9 targeted deletions that restore the reading frame could convert DMD genotypes into BMD-like genotypes and potentially treat this disease. Previously, this strategy has been demonstrated in vitro with zinc finger nucleases, TALENs, and S. pyogenes Cas9 leading to restoration of dystrophin expression in DMD patient myoblasts. However, these genome-editing enzymes are limited by the difficulty of delivering large transgenes with viral vectors in vivo. Alternatively, the smaller Cas9 ortholog from S. aureus (SaCas9) can be packaged with paired sgRNAs in an all-in-one AAV vector for in vivo gene therapy. Recently, three groups have demonstrated that AAV-SaCas9 can mediate targeted deletions in mdx mice and restore dystrophin expression. As a first step towards developing a genome editing therapeutic for DMD, we conducted an efficiency screen to identify highly active paired sgRNAs for targeted deletion of exon 51 of the dystrophin gene. First, a sensitive, digital droplet PCR assay to quantify exon 51 deletion was validated with DMD patient samples. An initial pilot study of 15 sgRNAs identified a pair (01+09) that mediated 18% Exon 51 deletion in HEK293T cells three days post-transfection. Next, from the set of 10,553 21-nt SaCas9 guides targeting human DMD introns 50 and 51, we selected 53 sgRNAs (675 pairs) that met the following filtering requirements: an endogenous 5’ G, a 3’ T in the NNGRR(T) PAM, cross-reactivity with the non-human primate (NHP) genome, and no off-by-1 or off-by-2 mismatch sites in the human genome. These filters were selected to improve U6 promoter expression, improve SaCas9 cleavage efficiency, enable pre-clinical animal model studies, and minimize off-target editing concerns, respectively. Notably, the NHP cross-reactivity requirement skewed the targeted deletion lengths towards larger sizes, all greater than 12.4kb. In order to test smaller deletions, an additional 174 pairs of human-only sgRNAs were designed for deletion lengths of 0.8-14kb. From the 850 guide pairs screened, 78 pairs with a deletion efficiency Z-score >1.5 were selected for follow-up validation. Interestingly, a few individual sgRNAs consistently appeared among the top performing pairs, regardless of whether they were paired with a generally less effective partner sgRNA. The top hit (guides 68+84), a human-only guide pair with a deletion size of 2.3kb, demonstrated a reproducible deletion efficiency of 32%. The 6 best human-only guide pairs and the 6 best NHP cross-reactive guide pairs have been cloned together with SaCas9 in all-in-one AAV vectors for testing in ex vivo and in vivo models of DMD.

P169 – 2697: A novel clinical trial design to evaluate the efficacy and safety of two exon-skipping drugs, SRP-4045 and SRP-4053, in a master protocol for patients with Duchenne muscular dystrophy (DMD)

Background DMD, an X-linked myopathy caused by mutations in the DMD gene, results in the inability to produce functional dystrophin, an essential protein for muscle fiber function. Exon-skipping phosphorodiamidate morpholino oligomers (PMOs) direct alternative splicing of dystrophin pre-mRNA, restoring the mRNA reading frame and enabling translation of an internally truncated yet functional dystrophin protein. SRP-4045 and SRP-4053 are PMOs intended to treat patients with mutations amenable to exon 45- and 53-skipping (16% of total DMD population). Methods A double-blind, placebo-controlled, multi-center, 48-week study is planned to evaluate the effects of SRP-4045 and SRP-4053 on ambulation, endurance and muscle function measured by the 6-Minute Walk Test (6MWT), and secondarily to evaluate restoration of dystrophin production and effect on pulmonary function tests (PFT). Patients (n=99) will be randomized by genotype in a double-blind fashion in 2:1 ratio, combined active-treatment (SRP-4045 or SRP-4053) to placebo. All patients will undergo a muscle biopsy at baseline and at either Week 24 or 48. Efficacy assessments will include 6MWT, PFT, NSAA, muscle strength, leg muscle MRI/MRS, PK, and dystrophin restoration profile. Safety assessments will include AE, ECG, ECHO, and safety laboratory tests. The primary analysis of clinical outcomes will be based on the combined population of patients who receive either drug compared to the placebo population. Discussion The study design highlights a novel and unique approach of combining the two exon-skipping drugs (SRP-4045 & SRP-4053) within the same trial, which is important as many DMD mutations have increasingly small patient-populations making it infeasible to power individual clinical studies. This approach may be the basis for approval of this class of chemistry for the rarest DMD genotypes by combining the patient-population to enable the enrollment of less prevalent genotypes while maintaining the statistical power to provide confidence in study results.

The NorthStar Ambulatory Assessment in Duchenne muscular dystrophy: considerations for the design of clinical trials

ObjectiveWith the emergence of experimental therapies for Duchenne muscular dystrophy (DMD), it is fundamental to understand the natural history of this disorder to properly design clinical trials. The aims of...

Dystrophin rescue by trans -splicing: a strategy for DMD genotypes not eligible for exon skipping approaches

RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre–trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations.

S.P.62 Ocular characteristics of Duchenne Muscular Dystrophy

<h2>Abstract</h2> Electroretinogram (ERG) abnormalities have been described in Duchenne Muscular Dystrophy (DMD). Short isoforms of dystrophin are highly expressed in retina. We sought to expand the retinal phenotypes associated with different DMD genotypes. We studied the ocular characteristics of 16 DMD boys, mean age 9.5years (range 4–15years), glucocorticosteroid (GC) treated, with a range of genotypes: six had mutations at the 5′ end of the gene involving exons 3–13, predicted to affect the full length dystrophin Dp427, seven patients exons 44–57, affecting Dp427 and Dp260, and three patients at the 3′ end, in exon 70, affecting expression of Dp427, Dp260 and Dp71. Visual acuity, colour vision, intra-ocular pressure measures (IOP) and full field ganzfeld flash ISCEV Electroretinograms (ERGs) were assessed. Patients had normal visual acuity, one boy had a red-green colour vision defect, and two had raised IOP due to steroid use. ‘Negative' scotopic ERGs with reduced a:b-wave amplitude ratios were recorded in all patients with mutations involving exons 44–57, one patient with a 8–13 deletion and two patients with the 3′ mutation involving exon 70. The scotopic oscillatory potential (OP2) was reduced in all boys with mutations of exons 44–57, and in one with 8–13. The photopic OP2 was reduced in patients with deletions upstream of exon 30, but preserved in those patients in whom scotopic OP2 was attenuated. Photopic ERG a:b-wave amplitude ratios were reduced across the cohort, including those with 5′ mutations in whom scotopic a:b ratios had been normal. These data show a strong association of mutations downstream of exon 30, the promoter region for Dp260, with abnormal scotopic ERGs. In contrast photopic ERG abnormalities were widespread across all genotypes which may relate to the greater proportion of Dp427, compared with other isoforms, at the synapse of cones to cone on-bipolar cells. Our clinical evaluation also reminds us to monitor IOP during GC treatment.

Detection of long-term progression of myocardial fibrosis in Duchenne muscular dystrophy in an affected family: A cardiovascular magnetic resonance study

Background Detection of myocardial fibrosis and left ventricular dysfunction in Duchenne muscular dystrophy (DMD) is the corner stone for further therapeutic studies. Little is known about the ability of cardiac magnetic resonance imaging (CMR) to evaluate progression of myocardial fibrosis. Aim of our study was to provide CMR data in a previously genotyped DMD family and to evaluate whether progression of myocardial fibrosis could be visualized. Methods and results DMD genotypes were available in 14 family members. CMR was performed in 4/5 carrier females, in 2/2 affected males and in one healthy family member with normal genotype. Functional images and late gadolinium enhanced (LGE) images in contiguous short-axis orientation were acquired at baseline and follow-up of 1231 days CMR examination could be repeated in three carrier females, in one affected male and in the healthy subject previously scanned. Mean decrease of left ventricular ejection fraction during the follow-up period was 10.5 ± 11.0%, mean progression of LGE volume 11.7 ± 9.5%. Conclusions Myocardial fibrosis seems to occur prior to global left ventricular dysfunction in DMD diseased males and carrier females. CMR could be used to evaluate progression of myocardial fibrosis and left ventricular function and may thus serve as an important diagnostic tool in the evaluation of therapeutical options in DMD.