Myodys, a full-length dystrophin plasmid vector for Duchenne and Becker muscular dystrophy gene therapy

Transgene SA is developing Myodys, a non-viral plasmid gene therapy for the potential treatment of Duchenne muscular dystrophy and Becker muscular dystrophy. Phase I clinical trials have been completed, and a phase II clinical trial is planned.     

The future of Duchenne muscular dystrophy gene therapy: shrinking the dystrophin gene

Duchenne muscular dystrophy is a debilitating muscle-wasting disease caused by mutations in the dystrophin gene - one of the largest genes identified thus far - and which ultimately results in premature death. With no current treatment available, the hopes of many sufferers lie in the establishment of an effective gene therapy. The adeno-associated virus is now emerging as a premium gene transfer vector eliciting minimal immune response from the host and allowing for long-term gene expression. It is the scope of this review to examine the recent efforts that have been made to develop ultra-truncated versions of the dystrophin gene that retain functionality, yet can still be cloned into recombinant adeno-associated viral vectors and other low-capacity vector systems.     

杜氏肌营养不良与贝氏肌营养不良常规MRI的对比研究

目的 探讨常规MRI鉴别诊断杜氏肌营养不良（DMD）和贝氏肌营养不良（BMD）的价值。方法 回顾性分析经临床确诊为抗肌萎缩蛋白病697例患者的臀部及大腿肌肉影像学资料,比较DMD与BMD影像学征象的异同。结果 DMD与BMD患者肌肉脂肪浸润的分布规律一致,DMD总体脂肪浸润程度高于BMD（P=0.034）,且以臀大肌、股直肌、缝匠肌为著;肌肉总体水肿程度差异无统计学意义（P=0.065）,但DMD大腿后群肌肉及缝匠肌的水肿明显高于BMD。DMD与BMD患者肌肉萎缩及肥大的选择性受累规律一致,BMD股外侧肌、股中间肌、股内侧肌、半膜肌及股二头肌长头萎缩频率明显高于DMD（P<0.05）;DMD与BMD患者肌肉的肥大改变差异无统计学意义（P>0.05）。结论 常规MRI可鉴别诊断DMD与BMD。     

A comparison of swallowing dysfunction in Becker muscular dystrophy and Duchenne muscular dystrophy

Purpose: Swallowing dysfunction has been reported in Duchenne muscular dystrophy (DMD), but has not been studied in Becker muscular dystrophy (BMD). The aims of this study were to report the characteristics of swallowing dysfunction in BMD compared with DMD.

Materials and methods: The study participants were 18 patients with BMD and 18 patients with DMD. All the patients were examined using videofluorography during swallowing of 5?mL of fluid. The penetration–aspiration scale (P–A scale) and the videofluorographic dysphagia scale (VDS) were used to evaluate dysphagia.

Results: Swinyard functional ability stage was not significantly different between the BMD and DMD groups. Rate of aspiration, P–A scale score, and total VDS score did not differ across groups, but the VDS item score for laryngeal elevation was lower in the BMD group than in the DMD group (median scores 4.5 and 9, respectively; p?r?=?0.78, p?Conclusion: Patients with BMD have swallowing problems similar to those observed in patients with DMD when matched according to physical functional status. These patients should be evaluated and followed-up for the duration of their disease.

• Implications for rehabiliation

• Dysphagia is one of the most critical problems in patients with progressive neuromuscular disease but dysphagia in patients with Becker muscular dystrophy (BMD) was not well known.

• Eighteen patients with BMD and 18 patients with Duchenne muscular dystrophy were examined with videofluorography.

• Patients with BMD have swallowing problems similar to those observed in patients with DMD.

     

假肥大型肌营养不良症产前基因诊断的临床应用

目的 建立规范的假肥大型肌营养不良症(DMD/BMD)产前基因诊断程序。方法 利用DMD基因内18个缺失热点的PCR引物,6个DMD基因内(CA)n短重复序列(STR)引物以及性别决定基因SRY引物,多重聚合酶链反应检测羊水胎儿脱落细胞、先证者和双亲外周血有核细胞基因组DMD基因,通过胎儿性别确定、基因缺失检测和基因连锁分析确定胎儿DMD基因状态。结果 在4例先证者基因缺失家系的产前诊断中,2例男胎DMD基因缺失与先证者相同,1例女胎为基因缺失携带者,1例男胎未发现缺失;在4例有2个或2个以上患者又先证者未检出缺失的家系产前诊断中,发现获得风险染色体的男胎与女胎各1例,余1例女胎和1例男胎均未携带风险染色体。检测结果的可靠性经流产胚胎和已出生胎儿DNA分析、DMD临床症状监测得到部分证实。结论 胎儿性别基因诊断、DMD基因缺失检测结合基因连锁分析的方法,在严格控制检测范围、规范的检测程序和有效的质量控制下,能准确地对DMD／BMD进行产前基因诊断,是目前预防患儿出生最有效的实验室检测方法之一。     

应用三联PCR技术产前诊断假性肥大型进行性肌营养不良的研究

目的通过对两例散发的BMD家系中的先证者和胎儿进行DMD基因和SRY基因分析,拟建立一种快速、准确、适用于临床的DMD/BMD的产前基因诊断方法。方法提取羊水细胞基因组DNA,通过检测SRY基因鉴定胎儿性别,以多重PCR方法对DMD基因缺失热区的18个外显子进行基因缺失的筛查,结合4对STR引物(内含子44、45、49、50)进行短串联重复序列多态性连锁分析(STR-PCR),分析结果经DNA测序验证。结果 SRY基因分析结果显示家系1胎儿为女性,家系2胎儿为男性,与超声诊断结果一致。两例BMD先证者均未检测到18个外显子的缺失,两家系的母亲及家系1的女性胎儿均为BMD基因STR-49位点杂合子,家系2的男性胎儿为BMD。同时发现了STR45、STR50的非常见基因型。结论应用SRY基因检测胎儿性别及多重PCR方法对DMD基因外显子缺失的检测,结合STR-PCR技术检出非缺失型携带者并行产前诊断,此三联PCR方法具有快速、简便、直观可靠的特点,特异性高,实验可操作性强,适用于临床推广应用。     

Reliability of testing measures in Duchenne or Becker muscular dystrophy

In a multiinstitutional collaborative study, we ascertained the interevaluator and intraevaluator reliability of six physical therapists who performed assessment measures on 36 boys (11.7 +/- 3.9 years) with Duchenne or Becker muscular dystrophy. Upper and lower extremities were evaluated by manual muscle testing for function, range of motion, and strength. The data were analyzed using intraclass correlation coefficients (ICCs). For the interevaluator phase, ICCs were as follows: average muscle strength, .90; range of motion, .76; and upper extremity functional performance, .58. For the intraevaluator phase, corresponding ICCs were .80 to .96; .33 to .97; .34 to 1.00. Our results confirm and extend observations by others that these assessment measures are sufficiently reliable for use in a multiinstitutional collaborative effort. Such results can be used to design clinical trials that have sufficient statistical power to detect changes in the rate of disease progression. Investigators planning clinical trials in a multiinstitutional collaborative setting should first standardize the assessment methods, provide evaluator training, and document reliability.     

Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.     

Duchenne型肌营养不良症的治疗进展

Duchenne型肌营养不良症(DMD)是一种X连锁隐性遗传的致死性肌病,一般6岁左右发病,9~14岁渐至不能行走,多于20岁左右死于心、肺功能衰竭。目前DMD的治疗方法匮乏,本文对DMD治疗进展进行综述。     

Current understanding and management of dilated cardiomyopathy in Duchenne and Becker muscular dystrophy

Purpose: To review the current understanding of the pathophysiology of dilated cardiomyopathy (DCM) in patients with Duchenne and Becker muscular dystrophies, assessment of cardiac dysfunction for these patients, and the recommended pharmacological treatment options and ongoing research directions.
Data sources: Reviews and original research articles from scholarly journals and books.
Conclusions: Duchenne and Becker muscular dystrophies are debilitating neuromuscular disorders, both caused by mutations in the dystrophin gene. Most patients develop DCM as part of the disease course; in fact, DCM is the leading cause of death among these patients. Cardiac surveillance, including routine monitoring of electrocardiograms, echocardiograms, and appropriate blood biomarkers, may detect early DCM development. Although previous studies have shown that early administration of cardiac medications may delay the development of DCM, current standard of care does not emphasize cardiac surveillance and timely treatment. This, in turn, limits clinicians, including advanced practice nurses, to be optimally engaged in providing the most aggressive and proactive patient care.
Implications for practice: Implementing a routine cardiac assessment and timely pharmacological treatment in primary or specialty care settings is highlighted as an important step to optimize cardiac health among patients with Duchenne and Becker muscular dystrophies.     

Immunohistochemical analysis of dystrophin-associated proteins in Becker/Duchenne muscular dystrophy with huge in-frame deletions in the NH2-terminal and rod domains of dystrophin.

The absence of dystrophin causes the drastic reduction of the dystrophin-associated proteins (DAPs) in the sarcolemma and the loss of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in Duchenne muscular dystrophy (DMD) skeletal muscle. Here, we report a mild reduction of the DAPs in the unique Becker muscular dystrophy patients with huge deletions in the rod domain of dystrophin and a moderate reduction of the DAPs in patients with huge deletions that involve both the NH2-terminal and rod domains of dystrophin. The phenotype of the latter patients was more severe than that of the former. In both cases, however, the reduction in the DAPs was milder than in typical DMD patients or DMD patients lacking the COOH-terminal domains of dystrophin. Our results suggest that (a) the NH2-terminal and rod domains of dystrophin may not be essential for the interaction with the sarcolemmal glycoprotein complex; and (b) defects in the actin binding activity of dystrophin may cause disruption of the anchorage of the dystrophin-glycoprotein complex to the subsarcolemmal cytoskeleton, which may render muscle fibers susceptible to degeneration.     

应用多重聚合酶链反应与聚丙烯酰胺凝胶电泳提高肌营养不良基因缺失检出率

目的 检测假肥大型肌营养不良（DMD／BMD）基因缺失及提高缺失检出率。方法 采用26对引物和多重聚合酶链反应（mPCR）及聚丙烯酰胺凝胶（PAGE）电泳技术，对74例DMD／BMD患者进行基因检测分析。结果 共检出42例基因缺失病人，缺失率为56.7%，主要分布于中央缺失热区和5′端缺失热区，其中以48号和49号外显子缺失最为多见，这两种技术结合应用，可检出常规方法未能检出的缺失。结论 基因缺失主要分布于44－52号外显子和1－19与外显子两个缺失热区。采用26对引物mPCR技术及PAGE电泳技术可有效提高基因缺失检出率。     

Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.

Recent molecular studies have shown that in a patient with Duchenne muscular dystrophy (DMD) Kobe, the size of exon 19 of the dystrophin gene was reduced to 36 bp due to the deletion of 52 bp out of 88 bp of the exon. The consensus sequences at the 5' and 3' splice sites of exon 19 were unaltered (Matsuo, M., et al. 1990. Biochem. Biophys. Res. Commun. 170:963-967). To further elucidate the molecular nature of the defect, we examined the primary structure of cytoplasmic dystrophin mRNA of the DMD Kobe patient across the junctions of exons 18, 19, and 20 by gel electrophoresis and sequencing of polymerase chain reaction-amplified cDNA. The mRNA coding for dystrophin was reverse transcribed using random primers, and the cDNA was then enzymatically amplified in vitro. The targeted fragment was smaller than expected from the genomic DNA analysis. By sequencing of the amplified product, we found that exon 18 was joined directly to exon 20, so that exon 19 was completely absent, suggesting that this exon was skipped during processing of the dystrophin mRNA precursor. All other bases in the amplified product were unaltered. Therefore, the data strongly suggest that the internal exon deletion generates an abnormally spliced mRNA in which the sequence of exon 18 is joined to the sequence of exon 20. We propose that the deletion is responsible for abnormal processing of the DMD Kobe allele. This finding has important implications regarding the determinants of a functional splice site.     

Immune responses to dystropin: implications for gene therapy of Duchenne muscular dystrophy

Introduction of dystrophin by gene transfer into the dystrophic muscles of Duchenne muscular dystrophy (DMD) patients has the possibility of triggering an immune response as many patients will not have been exposed to some (or all) of the epitopes of dystrophin. This could in turn lead to cytotoxic destruction of transfected muscle fibres. We assessed such concerns in the dystrophin-deficient mdx mouse using plasmid DNA as the gene transfer system. This avoids complications associated with the administration of viral proteins. Gene transfer of cDNAs encoding mouse full-length or a truncated minidystrophin did not evoke either a humoral or cytotoxic immune response. Mdx mice may be tolerant due to the presence of rare 'revertant' dystrophin-positive fibres in their skeletal muscles. In contrast, gene transfer of human full-length or minidystrophin provoked both humoral and cytotoxic responses leading to destruction of the transfected fibres. These experiments demonstrate the potential risk of deleterious effects following gene therapy in DMD patients and lead us to suggest that patients enrolled in gene therapy trials should ideally have small, preferably point, mutations and evidence of 'revertant' dystrophin-positive muscle fibres.     

Quantitative and qualitative alterations of dystrophin are expressed in muscle cell cultures of Xp21 muscular dystrophy patients (Duchenne and Becker type)

The authors studied skeletal muscle cell cultures from four control subjects, two patients with Duchenne muscular dystrophy, two Duchenne carriers and three patients with Becker muscular dystrophy with different phenotypes. Western blotting was performed on well-differentiated myotubes and compared with the results obtained in muscle tissue. In all cultures the band of dystrophin closely corresponded to the one observed in muscle tissue: both quantitative and qualitative defects were observed. This confirms the early expression of the Xp21 gene defect in uninnervated muscle cultures and supports the usefulness of muscle cultures both in diagnostic procedure and as a model to study the disease.     

Potential of oligonucleotide-mediated exon-skipping therapy for Duchenne muscular dystrophy

Many of the mutations associated with Duchenne muscular dystrophy can potentially be rescued by exon-skipping therapy, targeting selected exons of prespliced mRNA for the dystrophin gene with antisense oligonucleotides, thereby restoring reading frames. The recent development of antisense oligonucleotides with higher stability and lower toxicity, such as morpholinos, has made it possible to restore dystrophin efficiently in dystrophic mice in vivo with no obvious side effects. There seems little doubt that such exon-skipping therapy is destined to proceed to the clinical application stage in patients with Duchenne muscular dystrophy. One of the remaining issues to be addressed is the skipping of multiple exons because such multi-exon skipping therapy could expand the potential patient target population to include 80% of those with duplication mutations and 90% of those with deletion mutations. At present, this multi-exon skipping strategy is being investigated in dystrophic dogs as well as dystrophic mice. There are several challenges that still need to be overcome, including the low uptake of antisense oligonucleotides into the heart and the need to design efficient, nontoxic, cost-effective oligonucleotides. This review summarizes recent progress in exon-skipping therapy and discusses future perspectives with regard to human clinical trials.