Search

• DMD is caused by DMD gene mutations that result in a lack of dystrophin protein. • BMD is also caused by DMD mutations, but symptoms are milder compared with DMD. • We present a case study of a patient with BMD and his affected relatives. • The patient could walk at age 61 years, despite 46% of his DMD gene being missing. • These findings informed minigene constructs, a promising therapy option for DMD. Duchenne muscular dystrophy is a neuromuscular disease caused by DMD gene mutations that result in an absence of functional dystrophin protein. Patients with Duchenne experience progressive muscle weakness, are typically wheelchair dependent by their early teens, and develop respiratory and cardiac complications that lead to death in their twenties or thirties. Becker muscular dystrophy is also caused by DMD gene mutations, but symptoms are less severe and progression is slower compared with Duchenne. We describe a case study of a patient with Becker muscular dystrophy who was still ambulant at age 61 years and had a milder phenotype than Duchenne, despite 46% of his DMD gene being missing. His affected relatives had similarly mild phenotypes and clinical courses. These data guided the understanding of the criticality of various regions of dystrophin and informed the development of micro-dystrophin constructs to compensate for the absence of functional dystrophin in Duchenne. [ABSTRACT FROM AUTHOR]

Research advances made in the last decade and half in characterizing the mechanisms underlying the development of various forms of muscular dystrophy augurs well for optimism in managing and treating muscular dystrophy. Discovery of dystrophin and its molecular implications in Duchenne type and Becker muscular dystrophies are discussed.

The fatal muscle wasting disease Duchenne muscular dystrophy is caused by the lack of dystrophin in muscle membranes. Dystrophin deficiency, however, does not affect mice. A study on mice with dystrophin and dystrophin-related protein utrophin deficiency reveal ultrastructural neuromuscular and myotendinous junction abnormalities that causes premature death. Results indicate that dystrophin and utrophin have complementary functions in developmental muscular pathways.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.05.002 Byline: Kevin Talbot (1), Kay E. Davies (1) Abstract: Spinal muscular atrophy (SMA) is caused by a drastic reduction in the ubiquitously expressed SMN protein, which is critical for the correct assembly of the snRNP complexes required for RNA splicing. However, it is unclear why loss of SMN and altered snRNP assembly only seem to affect motor neurons. Reporting in this issue, challenge prior assumptions about the housekeeping function of SMN and demonstrate that loss of SMN leads to highly tissue-specific effects on splicing. Author Affiliation: (1) MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.12.010 Byline: Kay E. Davies (1), Miranda D. Grounds (2) Abstract: There is currently no effective treatment for the devastating muscle-wasting disease Duchenne muscular dystrophy (DMD). Cossu and colleagues report in a recent Nature paper that transplantation of mesoangioblast stem cells may hold promise for treating DMD. Further studies are required to fully evaluate the clinical potential of these blood-vessel-associated stem cells. Author Affiliation: (1) MRC Functional Genetics Unit, University of Oxford, Oxford, UK (2) School of Anatomy & Human Biology, the University of Western Australia, Australia

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of dystrophin, essential for muscle fibre integrity. Despite extensive pre-clinical studies, development of an effective treatment has proved challenging. More recently, significant progress has been made with the first drug approval using a genetic approach and the application of pharmacological agents which slow the progression of the disease. Drug development for DMD has mainly used two strategies: (1) the restoration of dystrophin expression or the expression of the compensatory utrophin protein as an efficient surrogate, and (2) the mitigation of secondary downstream pathological mechanisms. This review details current most promising pharmacological approaches and clinical trials aiming to tackle the pathogenesis of this multifaceted disorder. [ABSTRACT FROM AUTHOR]

Oxidative stress (OS) and mitochondrial dysfunction are implicated in neurodegenerative disease, suggesting that antioxidant defence systems are critical for cell survival in the central nervous system (CNS). Oxidation resistance 1 (OXR1) can protect against OS in cellular and mouse models of amyotrophic lateral sclerosis (ALS) when over-expressed, whereas deletion of Oxr1 in mice causes neurodegeneration. OXR1 has emerged therefore as an essential antioxidant protein that controls the susceptibility of neurons to OS. It has been suggested that OXR1 is localised to mitochondria, yet the functional significance of this has not been investigated in the context of neuronal cell death. In order to characterise the role of Oxr1 in mitochondria, we investigated its sub-mitochondrial localisation and demonstrate that specific isoforms are associated with the outer mitochondrial membrane, while the full-length Oxr1 protein is predominately cytoplasmic. Interestingly, cytoplamsic over-expression of these mitochondrially-localised isoforms was still able to protect against OS-induced cell death and prevent rotenone-induced mitochondrial morphological changes. To study the consequences of Oxr1 deletion in vivo , we utilised the bella ataxic mouse mutant. We were unable to identify defects in mitochondrial metabolism in primary cerebellar granule cells (GCs) from bella mice, however a reduction in mitochondrial length was observed in mutant GCs compared to those from wild-type. Furthermore, screening a panel of proteins that regulate mitochondrial morphology in bella GCs revealed de-regulation of phospho-Drp1(Ser616), a key mitochondrial fission regulatory factor. Our data provide new insights into the function of Oxr1, revealing that specific isoforms of this novel antioxidant protein are associated with mitochondria and that the modulation of mitochondrial morphology may be an important feature of its protective function. These results have important implications for the potential use of OXR1 in future antioxidant therapies. [ABSTRACT FROM AUTHOR]

Epithelia are positioned at a critical interface to prevent invasion by microorganisms from the environment. Pattern recognition receptors are important components of innate immunity because of their ability to interact with specific microbe-associated structures and initiate immune responses. Several distinct groups of receptors have been recognized. One of these, the scavenger receptors, has been classified into at least eight separate classes. The class A scavenger receptors are characterized by the presence of a collagen-like domain and include macrophage scavenger receptor type A (SR-A1 I/II, SCARA1) and MARCO (SCARA2). These receptors are known to make important contributions to host defense. Here, we identify a novel murine scavenger receptor, SCARA5, which has a structure typical of this class. The cDNA encodes 491 amino acids, which predict a type II protein that contains C-terminal intracellular, transmembrane, extracellular spacer, collagenous, and N-terminal scavenger receptor cysteine rich domains. Expression in Chinese hamster ovary cells confirmed that the receptor assembles as a homotrimer and is expressed at the plasma membrane. SCARA5-transfected cells bound Escherichia coli and Staphylococcus aureus, but not zymosan, in a polyanionic-inhibitable manner. Unlike other class A scavenger receptors, the receptor was unable to endocytose acetylated or oxidized low density lipoprotein. Quantitative RT-PCR and in situ hybridization demonstrate SCARA5 has a tissue and cellular distribution unique among class A scavenger receptors. Because of the restriction of SCARA5 transcripts to populations of epithelial cells, we propose that this receptor may play important roles in the innate immune activities of these cells. [ABSTRACT FROM AUTHOR]

Oxidative stress is a pathological feature of many neurological disorders; therefore, utilizing proteins that are protective against such cellular insults is a potentially valuable therapeutic approach. Oxidation resistance 1 (OXR1) has been shown previously to be critical for oxidative stress resistance in neuronal cells; deletion of this gene causes neurodegeneration in mice, yet conversely, overexpression of OXR1 is protective in cellular and mouse models of amyotrophic lateral sclerosis. However, the molecular mechanisms involved are unclear. OXR1 contains the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) domain, a motif present in a family of proteins including TBC1 domain family member 24 (TBC1D24), a protein mutated in a range of disorders characterized by seizures, hearing loss, and neurodegeneration. The TLDc domain is highly conserved across species, although the structure-function relationship is unknown. To understand the role of this domain in the stress response, we carried out systematic analysis of all mammalian TLDc domain-containing proteins, investigating their expression and neuroprotective properties in parallel. In addition, we performed a detailed structural and functional study of this domain in which we identified key residues required for its activity. Finally, we present a new mouse insertional mutant of Oxr1, confirming that specific disruption of the TLDc domain in vivo is sufficient to cause neurodegeneration. Our data demonstrate that the integrity of the TLDc domain is essential for conferring neuroprotection, an important step in understanding the functional significance of all TLDc domain-containing proteins in the cellular stress response and disease. [ABSTRACT FROM AUTHOR]

[Display omitted] A therapeutic approach that holds the potential to treat all Duchenne muscular dystrophy (DMD) patient populations is utrophin modulation. Ezutromid, a first generation utrophin modulator which was later found to act via antagonism of the arylhydrocarbon receptor, progressed to Phase 2 clinical trials. Although interim data showed target engagement and functional improvements, ezutromid ultimately failed to meet its clinical endpoints. We recently described the identification of a new class of hydrazide utrophin modulators which has a different mechanism of action to ezutromid. In this study we report our early optimisation studies on this hydrazide series. The new analogues had significantly improved potency in cell-based assays, increased sp3 character and reduced lipophilicity, which also improved their physicochemical properties. A representative new analogue combining these attributes increased utrophin protein in dystrophic mouse cells showing it can be used as a chemical tool to reveal new insights regarding utrophin upregulation as a strategy for DMD therapeutic intervention. [ABSTRACT FROM AUTHOR]

Mucolipidosis II (MLII) is a lysosomal storage disorder caused by loss of N-acetylglucosamine-1-phosphotransferase, which tags lysosomal enzymes with a mannose 6-phosphate marker for transport to the lysosome. In MLII, the loss of this marker leads to deficiency of multiple enzymes and non-enzymatic proteins in the lysosome, leading to the storage of multiple substrates. Here we present a novel mouse model of MLII homozygous for a patient mutation in the GNPTAB gene. Whereas the current gene knock-out mouse model of MLII lacks some of the characteristic features of the human disease, our novel mouse model more fully recapitulates the human pathology, showing growth retardation, skeletal and facial abnormalities, increased circulating lysosomal enzymatic activities, intracellular lysosomal storage, and reduced life span. Importantly, MLII behavioral deficits are characterized for the first time, including impaired motor function and psychomotor retardation. Histological analysis of the brain revealed progressive neurodegeneration in the cerebellum with severe Purkinje cell loss as the underlying cause of the ataxic gait. In addition, based on the loss of Npc2 (Niemann-Pick type C 2) protein expression in the brain, the mice were treated with 2-hydroxypropyl-β-cyclodextrin, a drug previously reported to rescue Purkinje cell death in a mouse model of Niemann-Pick type C disease. No improvement in brain pathology was observed. This indicates that cerebellar degeneration is not primarily triggered by loss of Npc2 function. This study emphasizes the value of modeling MLII patient mutations to generate clinically relevant mouse mutants to elucidate the pathogenic molecular pathways of MLII and address their amenability to therapy. [ABSTRACT FROM AUTHOR]

Abstract: Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1G93A mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1G93A mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1G93A mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS. [Copyright &y& Elsevier]

In the context of future adeno-associated viral (AAV)-based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD but still faces personalized medicine challenges as different mutations found in DMD patients require skipping of different exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or 'hot-spot' in the central genomic region. In this study, we have developed 11 different U7 small-nuclear RNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of exons 45 to 55. We demonstrate that these constructs induce efficient exon skipping both in vitro in DMD patients' myoblasts and in vivo in human DMD (hDMD) mice and that they can be combined into a single vector to achieve a multi skipping of at least 3 exons. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using adeno-associated viral (AAV) vectors encoding multiple U7 small-nuclear RNAs (U7snRNAs), offering therefore very promising tools for clinical treatment of DMD. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a genetic disease affecting about one in every 3,500 boys. This X-linked pathology is due to the absence of dystrophin in muscle fibers. This lack of dystrophin leads to the progressive muscle degeneration that is often responsible for the death of the DMD patients during the third decade of their life. There are currently no curative treatments for this disease but different therapeutic approaches are being studied. Gene therapy consists of introducing a transgene coding for full-length or a truncated version of dystrophin complementary DNA (cDNA) in muscles, whereas pharmaceutical therapy includes the use of chemical/biochemical substances to restore dystrophin expression or alleviate the DMD phenotype. Over the past years, many potential drugs were explored. This led to several clinical trials for gentamicin and ataluren (PTC124) allowing stop codon read-through. An alternative approach is to induce the expression of an internally deleted, partially functional dystrophin protein through exon skipping. The vectors and the methods used in gene therapy have been continually improving in order to obtain greater encapsidation capacity and better transduction efficiency. The most promising experimental approaches using pharmaceutical and gene therapies are reviewed in this article. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. In most cases, the open-reading frame is disrupted which results in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and has recently been shown to correct the reading frame and restore dystrophin expression in vitro and in vivo. Specific exon skipping can be achieved using synthetic oligonucleotides or viral vectors encoding modified small nuclear RNAs (snRNAs), by masking important splicing sites. In this study, we demonstrate that enhanced exon skipping can be induced by a U7 snRNA carrying binding sites for the heterogeneous ribonucleoprotein A1 (hnRNPA1). In DMD patient cells, bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51, and thus restore dystrophin expression to near wild-type levels. Furthermore, we show the efficacy of these constructs in vivo in transgenic mice carrying the entire human DMD locus after intramuscular injection of adeno-associated virus (AAV) vectors encoding the bifunctional U7 snRNA. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications.Molecular Therapy (2009) 17 7, 1234–1240. doi:10.1038/mt.2009.113 [ABSTRACT FROM AUTHOR]

We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2- and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx × Utrn-/- mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx × Utrn-/- mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation. [ABSTRACT FROM AUTHOR]