Challenges to oligonucleotides-based therapeutics for Duchenne muscular dystrophy

Antisense oligonucleotides are short nucleic acids designed to bind to specific messenger RNAs in order to modulate splicing patterns or inhibit protein translation. As such, they represent promising therapeutic tools for many disorders and have been actively developed for more than 20 years as a form of molecular medicine. Although significant progress has been made in developing these agents as drugs, they are yet not recognized as effective therapeutics and several hurdles remain to be overcome. Within the last few years, however, the prospect of successful oligonucleotides-based therapies has moved a step closer, in particular for Duchenne muscular dystrophy. Clinical trials have recently been conducted for this myopathy, where exon skipping is being used to achieve therapeutic outcomes. In this review, the recent developments and clinical trials using antisense oligonucleotides for Duchenne muscular dystrophy are discussed, with emphasis on the challenges ahead for this type of therapy, especially with regards to delivery and regulatory issues

Search for the hero: an investigation into the sports heroes of British sports fans

This is an initial study into British sports fans’ heroes. A questionnaire was sent to 95 students (average age ¼ 19.75) to identify their sporting hero, the hero’s sport and nationality and the reasons for this choice. Football was the most common source of sports heroes, identified by 49% of participants with a sporting hero. The majority (60%, N ¼ 48) of heroes chosen by participants were British, with David Beckham the most popular choice. Differences were observed between the gender of participants, gender of hero chosen and the reasons for choosing the hero. The most common reason for selecting a hero was a personal trait rather than skill, while in the questionnaire a category of Local Affiliation was added to those suggested by previous work. It was concluded that to become a hero athletes should combine skill with devotion to family, charity work and a place in popular culture

Rescue of severely affected dystrophin/utrophin-deficient mice through scAAV-U7snRNA-mediated exon skipping

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 and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype

In vivo MRI Characterization of Progressive Cardiac Dysfunction in the mdx Mouse Model of Muscular Dystrophy

Aims The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice. Methods and Results Serial measurements of cardiac morphology and function were made in the same group of mdx mice and controls (housed in a non-SPF facility) using MRI at 1, 3, 6, 9 and 12 months after birth. Left ventricular (LV) and right ventricular (RV) systolic and diastolic function, response to dobutamine stress and myocardial fibrosis were assessed. RV dysfunction preceded LV dysfunction, with RV end systolic volumes increased and RV ejection fractions reduced at 3 months of age. LV ejection fractions were reduced at 12 months, compared with controls. An abnormal response to dobutamine stress was identified in the RV of mdx mice as early as 1 month. Late-gadolinium-enhanced MRI identified increased levels of myocardial fibrosis in 6, 9 and 12-month-old mdx mice, the extent of fibrosis correlating with the degree of cardiac remodeling and hypertrophy. Conclusions MRI could identify cardiac abnormalities in the RV of mdx mice as young as 1 month, and detected myocardial fibrosis at 6 months. We believe these to be the earliest MRI measurements of cardiac function reported for any mice, and the first use of late-gadolinium-enhancement in a mouse model of congenital cardiomyopathy. These techniques offer a sensitive and clinically relevant in vivo method for assessment of cardiomyopathy caused by muscular dystrophy and other diseases

A parametric physical model for the intracluster medium and its use in joint SZ/X-ray analyses of galaxy clusters

We present a parameterized model of the intra-cluster medium that is suitable for jointly analysing pointed observations of the Sunyaev-Zel'dovich (SZ) effect and X-ray emission in galaxy clusters. The model is based on assumptions of hydrostatic equilibrium, the Navarro, Frenk and White (NFW) model for the dark matter, and a softened power law profile for the gas entropy. We test this entropy-based model against high and low signal-to-noise mock observations of a relaxed and recently-merged cluster from N-body/hydrodynamic simulations, using Bayesian hyper-parameters to optimise the relative statistical weighting of the mock SZ and X-ray data. We find that it accurately reproduces both the global values of the cluster temperature, total mass and gas mass fraction (fgas), as well as the radial dependencies of these quantities outside of the core (r > kpc). For reference we also provide a comparison with results from the single isothermal beta model. We confirm previous results that the single isothermal beta model can result in significant biases in derived cluster properties.Comment: Published in MNRAS. 20 pages. 9 figure

Deletion of smn-1, the Caenorhabditis elegans ortholog of the spinal muscular atrophy gene, results in locomotor dysfunction and reduced lifespan

Spinal muscular atrophy is the most common genetic cause of infant mortality and is characterized by degeneration of lower motor neurons leading to muscle wasting. The causative gene has been identified as survival motor neuron (SMN). The invertebrate model organism Caenorhabditis elegans contains smn-1, the ortholog of human SMN. Caenorhabditis elegans smn-1 is expressed in various tissues including the nervous system and body wall muscle, and knockdown of smn-1 by RNA interference is embryonic lethal. Here we show that the smn-1(ok355) deletion, which removes most of smn-1 including the translation start site, produces a pleiotropic phenotype including late larval arrest, reduced lifespan, sterility as well as impaired locomotion and pharyngeal activity. Mutant nematodes develop to late larval stages due to maternal contribution of the smn-1 gene product that allows to study SMN-1 functions beyond embryogenesis. Neuronal, but not muscle-directed, expression of smn-1 partially rescues the smn-1(ok355) phenotype. Thus, the deletion mutant smn-1(ok355) provides a useful platform for functional analysis of an invertebrate ortholog of the human SMN protein

Quantum Inequalities and Singular Energy Densities

There has been much recent work on quantum inequalities to constrain negative energy. These are uncertainty principle-type restrictions on the magnitude and duration of negative energy densities or fluxes. We consider several examples of apparent failures of the quantum inequalities, which involve passage of an observer through regions where the negative energy density becomes singular. We argue that this type of situation requires one to formulate quantum inequalities using sampling functions with compact support. We discuss such inequalities, and argue that they remain valid even in the presence of singular energy densities.Comment: 18 pages, LaTex, 2 figures, uses eps

Plectin 1f scaffolding at the sarcolemma of dystrophic (mdx) muscle fibers through multiple interactions with β-dystroglycan

In skeletal muscle, the cytolinker plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5–180 residues), four of which (plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using plectin isoform–specific antibodies and isoform expression constructs, we show the differential regulation of plectin isoforms during myotube differentiation and their localization to different compartments of muscle fibers, identifying plectins 1 and 1f as sarcolemma-associated isoforms, whereas plectin 1d localizes exclusively to Z-disks. Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct binding of plectin to dystrophin (utrophin) and β-dystroglycan, the key components of the dystrophin–glycoprotein complex. We propose a model in which plectin acts as a universal mediator of desmin intermediate filament anchorage at the sarcolemma and Z-disks. It also explains the plectin phenotype observed in dystrophic skeletal muscle of mdx mice and Duchenne muscular dystrophy patients