Duchenne muscular dystrophy is a devastating disease that leads to progressive muscle loss and premature death. While medical management focuses mostly on symptomatic treatment, decades of research have resulted in first therapeutics able to restore the affected reading frame of dystrophin transcripts or induce synthesis of a truncated dystrophin protein from a vector, with other strategies based on gene therapy and cell signaling in preclinical or clinical development. Nevertheless, recent reports show that potentially therapeutic dystrophins can be immunogenic in patients. This raises the question of whether a dystrophin paralog, utrophin, could be a more suitable therapeutic protein. Here, we compare dystrophin and utrophin amino acid sequences and structures, combining published data with our extended in silico analyses. We then discuss these results in the context of therapeutic approaches for Duchenne muscular dystrophy. Specifically, we focus on strategies based on delivery of micro-dystrophin and micro-utrophin genes with recombinant adeno-associated viral vectors, exon skipping of the mutated dystrophin pre-mRNAs, reading through termination codons with small molecules that mask premature stop codons, dystrophin gene repair by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genetic engineering, and increasing utrophin levels. Our analyses highlight the importance of various dystrophin and utrophin domains in Duchenne muscular dystrophy treatment, providing insights into designing novel therapeutic compounds with improved efficacy and decreased immunoreactivity. While the necessary actin and β-dystroglycan binding sites are present in both proteins, important functional distinctions can be identified in these domains and some other parts of truncated dystrophins might need redesigning due to their potentially immunogenic qualities. Alternatively, therapies based on utrophins might provide a safer and more effective approach.

Duchenne muscular dystrophy is caused by mutations in the dystrophin-encoding DMD gene. While Duchenne is most commonly caused by large intragenic deletions that cause frameshift and complete loss of dystrophin expression, in-frame deletions in DMD can result in the expression of internally truncated dystrophin proteins and may be associated with a milder phenotype. In this study, we describe two individuals with large in-frame 5\' deletions (exon 3-23 and exon 3-28) that remove the majority of the N-terminal region, including part of the actin binding and central rod domains. Both patients had progressive muscle weakness during childhood but are observed to have a relatively mild disease course compared to typical Duchenne. We show that in muscle biopsies from both patients, truncated dystrophin is expressed at the sarcolemma. We have additionally developed a patient-specific fibroblast-derived cell model, which can be inducibly reprogrammed to form myotubes that largely recapitulate biopsy findings for the patient with the exon 3-23 deletion, providing a culture model for future investigation of this unusual case. We discuss these mutations in the context of previously reported 5\' in-frame DMD deletions and relevant animal models, and review the spectrum of phenotypes associated with these deletions.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular disorders that primarily affect boys due to an X-linked mutation in the DMD gene, resulting in reduced to near absence of dystrophin or expression of truncated forms of dystrophin. Some newer therapeutic interventions aim to increase sarcolemmal dystrophin expression, and accurate dystrophin quantification is critical for demonstrating pharmacodynamic relationships in preclinical studies and clinical trials. Current challenges with measuring dystrophin include the variation in protein expression within individual muscle fibers and across whole muscle samples, the presence of preexisting dystrophin-positive revertant fibers, and trace amounts of residual dystrophin. Immunofluorescence quantification of dystrophin can overcome many of these challenges, but manual quantification of protein expression may be complicated by variations in the collection of images, reproducible scoring of fluorescent intensity, and bias introduced by manual scoring of typically only a few high-power fields. This review highlights the pathology of DMD and BMD, discusses animal models of DMD and BMD, and describes dystrophin biomarker quantitation in DMD and BMD, with several image analysis approaches, including a new automated method that evaluates protein expression of individual muscle fibers.

The membrane cytoskeletal component dystrophin and its associated glycoproteins play a central role in the molecular pathogenesis of several muscular dystrophies, i.e. Duchenne/Becker muscular dystrophy, congenital muscular dystrophy and various forms of limb-girdle muscular dystrophy. Although the most frequent of these disorders, Duchenne muscular dystrophy, is mainly recognized as a disease of skeletal muscle fibers, pathophysiological changes also involve the heart and diaphragm, as well as the peripheral and central nervous system. Thus current research efforts into the elucidation of the molecular mechanisms underlying these genetic diseases are not only directed towards studying skeletal muscle necrosis but also investigate abnormalities of heart and brain dystrophin-glycoprotein complexes in cardiomyopathy and brain deficiencies associated with muscular dystrophy. Furthermore, many isoforms of dystrophin and dystrophin-associated components have been identified in various non-muscle tissues and their function(s) are mostly unknown. With respect to skeletal muscle fibers, the characterization of new dystrophin-associated proteins, such as dystrobrevin, sarcospan and the syntrophins, led to a modified model of the spatial configuration of the dystrophin-glycoprotein complex. However, it is generally accepted now that beta-dystroglycan forms the plasmalemma-spanning linkage between dystrophin and the laminin-binding protein alpha-dystroglycan and that this complex is associated with the sarcoglycan subcomplex of sarcolemmal glycoproteins.