Exon Skipping and Duchenne Muscular Dystrophy Therapy: Selection of the Most Active U1 snRNA Antisense Able to Induce Dystrophin Exon 51 Skipping

One promising approach for the gene therapy of Duchenne muscular dystrophy (DMD) is exon skipping. When thinking of possible intervention on human, it is very crucial to identify the most appropriate antisense sequences able to provide the highest possible skipping efficiency. In this article, we compared the exon 51 skipping activity of 10 different antisense molecules, raised against splice junctions and/or exonic splicing enhancers (ESEs), expressed as part of the U1 small nuclear RNA (snRNA). The effectiveness of each construct was tested in human DMD myoblasts carrying the deletion of exons 48–50, which can be treated with skipping of exon 51. Our results show that the highest skipping activity and dystrophin rescue is achieved upon expression of a U1 snRNA-derived antisense molecule targeting exon 51 splice sites in combination with an internal exon sequence. The efficacy of this molecule was further proven on an exon 45–50 deletion background, utilizing patient''s fibroblasts transdifferentiated into myoblasts. In this system, we showed that the selected antisense was able to produce 50% skipping of exon 51.     

Wheelchair seating for children with Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder which features progressive muscle wasting and weakness. Despite advances in treatment, the weakness of DMD will eventually necessitate a wheelchair for almost all children. The goal of wheelchair use is to maximize function and mobility while minimizing discomfort and postural abnormalities. Because of the large variation of patient symptoms and functional deficits, no single wheelchair would adequately serve the needs of all children with DMD. Unfortunately, little information to guide selection of equipment for children with DMD is available. This article discusses the decision-making processes regarding appropriate time to provide equipment, the evaluation of DMD clients, and reviews the numerous options in order to help prescribers, caregivers and clients design an appropriate wheelchair system.     

Laminin-111: A Potential Therapeutic Agent for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) still needs effective treatments, and myoblast transplantation (MT) is considered as an approach to repair damaged skeletal muscles. DMD is due to the complete loss of dystrophin from muscles. The lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins in the extracellular matrix. Laminin-111 is normally present in skeletal and cardiac muscles in mice and humans but only during embryonic development. In this study, we showed that intramuscular injection of laminin-111 increased muscle strength and resistance in mdx mice. We also used laminin-111 as a coadjuvant in MT, and we showed this protein decreased considerably the repetitive cycles of degeneration, inflammatory reaction, and regeneration. Moreover, MT is significantly improved. To explain the improvement, we confirmed with the same myoblast cell batch that laminin-111 improves proliferation and drastically increases migration in vitro. These results are extremely important because DMD could be treated only by the injection of a recombinant protein, a simple and safe therapy to prevent loss of muscle function. Moreover, the improvement in MT would be significant to treat the muscles of DMD patients who are already weak.     

杜氏肌营养不良症的临床表现与基因表型的关联

目的:分析杜氏肌营养不良症(DMD)患者的基因突变特点,探讨疾病的临床表现与基因型的关联。方法:回顾性分析52例DMD患者的临床表现和基因特点。结果:患者多表现为肢体无力、走路姿势异常,发育迟缓,部分患者伴智力下降,心脏功能减退等。多重链接探针依赖扩增技术发现基因检测无异常4例(7.69%),DMD基因缺失40例(76.9%),重复8例(15.3%)。基因突变发生在外显子45~55 22例(40.74%),2~19区域10例(19.23%)。48例基因异常患者中,符合阅读框架原则42例(87.5%)。结论:基因缺失的大小与临床症状的关系不大,病情严重程度关键取决于突变是否会破坏阅读框结构。基因突变越接近5'端对智力的影响越轻,越接近3'端对智力的影响越重。     

A Community-Engaged Approach to Quantifying Caregiver Preferences for the Benefits and Risks of Emerging Therapies for Duchenne Muscular Dystrophy

Background

There is growing agreement that regulators performing benefit–risk evaluations should take patients’ and caregivers’ preferences into consideration. The Patient-Focused Drug Development Initiative at the US Food and Drug Administration offers patients and caregivers an enhanced opportunity to contribute to regulatory processes by offering direct testimonials. This process may be advanced by providing scientific evidence regarding treatment preferences through engagement of a broad community of patients and caregivers.

Objective

In this article, we demonstrate a community-engaged approach to measure caregiver preferences for potential benefits and risks of emerging therapies for Duchenne muscular dystrophy (DMD).

Methods

An advocacy oversight team led the community-engaged study. Caregivers’ treatment preferences were measured by using best–worst scaling (BWS). Six relevant and understandable attributes describing potential benefits and risks of emerging DMD therapies were identified through engagement with advocates (n = 5), clinicians (n = 9), drug developers from pharmaceutical companies and academic centers (n = 11), and other stakeholders (n = 5). The attributes, each defined across 3 levels, included muscle function, life span, knowledge about the drug, nausea, risk of bleeds, and risk of arrhythmia. Cognitive interviewing with caregivers (n = 7) was used to refine terminology and assess acceptability of the BWS instrument. The study was implemented through an online survey of DMD caregivers, who were recruited in the United States through an advocacy group and snowball sampling. Caregivers were presented with 18 treatment profiles, identified via a main-effect orthogonal experimental design, in which the dependent variable was the respondents’ judgment as to the best and worst feature in each profile. Preference weights were estimated by calculating the relative number of times a feature was chosen as best and as worst, which were then used to estimate relative attribute importance.

Results

A total of 119 DMD caregivers completed the BWS instrument; they were predominately biological mothers (67.2%), married (89.9%), and white (91.6%). Treatment effect on muscle function was the most important among experimental attributes (28.7%), followed by risk of heart arrhythmia (22.4%) and risk of bleeding (21.2%). Having additional postapproval data was relatively the least important attribute (2.3%).

Conclusions

We present a model process for advocacy organizations aiming to promote patient-centered drug development. The community-engaged approach was successfully used to develop and implement a survey to measure caregiver preferences. Caregivers were willing to accept a serious risk when balanced with a noncurative treatment, even absent improvement in life span. These preferences should inform the Food and Drug Administration’s benefit–risk assessment of emerging DMD therapies. This study highlights the synergistic integration of traditional advocacy methods and scientific approach to quantify benefit–risk preferences.     

Cardiac and Respiratory Dysfunction in Duchenne Muscular Dystrophy and the Role of Second Messengers

Duchenne muscular dystrophy (DMD) affects young boys and is characterized by the absence of dystrophin, a large cytoskeletal protein present in skeletal and cardiac muscle cells and neurons. The heart and diaphragm become necrotic in DMD patients and animal models of DMD, resulting in cardiorespiratory failure as the leading cause of death. The major consequences of the absence of dystrophin are high levels of intracellular Ca²⁺ and the unbalanced production of NO that can finally trigger protein degradation and cell death. Cytoplasmic increase in Ca²⁺ concentration directly and indirectly triggers different processes such as necrosis, fibrosis, and activation of macrophages. The absence of the neuronal isoform of nitric oxide synthase (nNOS) and the overproduction of NO by the inducible isoform (iNOS) further increase the intracellular Ca²⁺ via a hypernitrosylation of the ryanodine receptor. NO overproduction, which further induces the expression of iNOS but decreases the expression of the endothelial isoform (eNOS), deregulates the muscle tissue blood flow creating an ischemic situation. The high levels of Ca²⁺ in dystrophic muscles and the ischemic state of the muscle tissue would culminate in a positive feedback loop. While efforts continue toward optimizing cardiac and respiratory care of DMD patients, both Ca²⁺ and NO in cardiac and respiratory muscle pathways have been shown to be important to the etiology of the disease. Understanding the mechanisms behind the fine regulation of Ca²⁺‐NO may be important for a noninterventional and noninvasive supportive approach to treat DMD patients, improving the quality of life and natural history of DMD patients.     

Glucose Metabolism as a Pre-clinical Biomarker for the Golden Retriever Model of Duchenne Muscular Dystrophy

Purpose

Metabolic dysfunction in Duchenne muscular dystrophy (DMD) is characterized by reduced glycolytic and oxidative enzymes, decreased and abnormal mitochondria, decreased ATP, and increased oxidative stress. We analyzed glucose metabolism as a potential disease biomarker in the genetically homologous golden retriever muscular dystrophy (GRMD) dog with molecular, biochemical, and in vivo imaging.

Procedures

Pelvic limb skeletal muscle and left ventricle tissue from the heart were analyzed by mRNA profiling, qPCR, western blotting, and immunofluorescence microscopy for the primary glucose transporter (GLUT4). Physiologic glucose handling was measured by fasting glucose tolerance test (GTT), insulin levels, and skeletal and cardiac positron emission tomography/X-ray computed tomography (PET/CT) using the glucose analog 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG).

Results

MRNA profiles showed decreased GLUT4 in the cranial sartorius (CS), vastus lateralis (VL), and long digital extensor (LDE) of GRMD vs. normal dogs. QPCR confirmed GLUT4 downregulation but increased hexokinase-1. GLUT4 protein levels were not different in the CS, VL, or left ventricle but increased in the LDE of GRMD vs. normal. Microscopy revealed diffuse membrane expression of GLUT4 in GRMD skeletal but not cardiac muscle. GTT showed higher basal glucose and insulin in GRMD but rapid tissue glucose uptake at 5 min post-dextrose injection in GRMD vs. normal/carrier dogs. PET/ CT with [¹⁸F]FDG and simultaneous insulin stimulation showed a significant increase (p?=?0.03) in mean standard uptake values (SUV) in GRMD skeletal muscle but not pelvic fat at 5 min post-[¹⁸F]FDG /insulin injection. Conversely, mean cardiac SUV was lower in GRMD than carrier/normal (p?<?0.01).

Conclusions

Altered glucose metabolism in skeletal and cardiac muscle of GRMD dogs can be monitored with molecular, biochemical, and in vivo imaging studies and potentially utilized as a biomarker for disease progression and therapeutic response.

     

High‐Frequency Ultrasound to Grade Disease Progression in Murine Models of Duchenne Muscular Dystrophy

Objective. This study used high‐frequency ultrasound (HFU) imaging to assess muscle damage noninvasively in a longitudinal study of 2 transgenic murine models of Duchenne muscular dystrophy (DMD): mdx, which has mutated cytoskeletal protein dystrophin; and udx, which has mutated dystrophin and lacks another cytoskeleton protein, utrophin. The mdx group was further subdivided into exercised and nonexercised subgroups to assess exercise‐induced damage. Methods. Muscle damage was assessed with HFU imaging (40 MHz) at biweekly intervals for 16 weeks. The assessment was based on the number of hyperechoic lesions, the lesion diameter, and muscle disorganization, giving a combined grade according to a 5‐point scale. Results. High‐frequency ultrasound discriminated the severity of muscle damage between wild‐type and transgenic models of DMD and between mdx and udx models. Qualitative comparisons of 3‐dimensional HFU images with serial histologic sections of the skeletal muscle showed the ability of ultrasound to accurately depict changes seen in the muscle architecture in vivo. Conclusions. High‐frequency ultrasound images soft tissue in mice at high contrast and spatial resolution, thereby showing that this microimaging modality has the capability to assess architectural changes in muscle fibers due to myotonic dystrophy–related diseases such as DMD.