Treatment Of Animal Models Research Articles

Depth-resolved measurement of mucosal microvascular blood content using low-coherence enhanced backscattering spectroscopy

Low-coherence enhanced backscattering (LEBS) spectroscopy is a light scattering technique which uses partial spatial coherence broadband illumination to interrogate the optical properties at sub-diffusion length scales. In this work, we present a post-processing technique which isolates the hemoglobin concentration at different depths within a sample using a single spectroscopic LEBS measurement with a fixed spatial coherence of illumination. We verify the method with scattering (spectralon reflectance standard and polystyrene microspheres) and absorbing (hemoglobin) phantoms. We then demonstrate the relevance of this method for quantifying hemoglobin content as a function of depth within biological tissue using the azoxymethane treated animal model of colorectal cancer.

Intravenous Immunoglobulin for the Treatment of Clostridium difficile Infection: A Review

Clostridium difficile infection (CDI) has increased sharply in incidence, mortality rate, and burden on the healthcare system over the past decade. Therefore, novel treatment modalities have been developed, including intravenous immunoglobulin (IVIG). The level of immune response to Clostridium difficile colonization is the major determinant of the magnitude and duration of clinical manifestations. This effect is mediated predominantly by serum IgG anti-toxin A antibodies. Based on this finding, anti-toxin A and B antibodies were successfully used in multiple in vitro and in vivo experimental settings to passively immunize hamsters in CDI models. In humans, IVIG was used as the source of those antibodies. Fifteen small, mostly retrospective and non-randomized reports documented IVIG's success in the treatment of protracted, recurrent, or severe CDI. Diarrhea resolution rates were higher in the former patient group, but the recurrence rates were similar. IVIG mechanism of action is neutralization of mainly toxin A through IgG anti-toxin A antibodies. Purified anti-toxin A and B antibodies were successfully used to decrease CDI recurrence rates among patients with no or one previous CDI episodes. In conclusion, the efficacy of IVIG for CDI treatment in animal models has been convincingly demonstrated. However, only few small non-randomized, mostly uncontrolled reports have been published on human subjects. A phase II trial results support the use of purified anti-toxin A and B antibodies to decrease CDI recurrence rates. Therefore, IVIG should currently only be used as adjunct therapy until results from large, randomized controlled trials are available.

The small chill: mild hypothermia for cardioprotection?

Reducing the heart's temperature by 2-5°C is a potent cardioprotective treatment in animal models of coronary artery occlusion. The anti-infarct benefit depends upon the target temperature and the time at which cooling is instituted. Protection primarily results from cooling during the ischaemic period, whereas cooling during reperfusion or beyond offers little protection. In animal studies, protection is proportional to both the depth and duration of cooling. An optimal cooling protocol must appreciably shorten the normothermic ischaemic time to effectively salvage myocardium. Patients presenting with acute myocardial infarction could be candidates for mild hypothermia since the current door-to-balloon time is typically 90 min. But they would have to be cooled quickly shortly after their arrival. Several strategies have been proposed for ultra-fast cooling, but most like liquid ventilation and pericardial perfusion are too invasive. More feasible strategies might include cutaneous cooling, peritoneal lavage with cold solutions, and endovascular cooling with intravenous thermodes. This last option has been investigated clinically, but the results have been disappointing possibly because the devices lacked capacity to cool the patient quickly or cooling was not implemented soon enough. The mechanism of hypothermia's protection has been assumed to be energy conservation. However, whereas deep hypothermia clearly preserves ATP, mild hypothermia has only a modest effect on ATP depletion during ischaemia. Some evidence suggests that intracellular signalling pathways might be responsible for the protection. It is unknown how cooling could trigger these pathways, but, if true, then it might be possible to duplicate cooling's protection pharmacologically.

Targeting Toll-like receptors on dendritic cells modifies the T H2 response to peanut allergens in vitro

Delivery of allergens with bacterial adjuvants has been shown to be a successful immunotherapeutic strategy for food allergy treatment in animal models. How microbial signals, acting through the innate immune system, reshape ongoing allergic responses is poorly understood. To investigate the contribution of Toll-like receptors (TLRs) in the response to bacterial adjuvants, we designed an in vitro system to characterize the effect of heat-killed Escherichia coli vector (HKE) on peanut-induced responses of dendritic cells (DCs) and T cells. Wild-type or TLR signaling-deficient bone marrow-derived DCs were pulsed with crude peanut extract (CPE) alone (50 microg/mL) in the presence of HKE (10(6)/mL). DC maturation was analyzed by means of flow cytometry. Treated DCs were cocultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4(+) T cells from sensitized mice. Cytokine production from DCs and T cells was measured by using Bioplex assays. Peanut-pulsed DCs induced the production of IL-4, IL-5, and IL-13, as well as IL-17 and IFN-gamma, from primed T cells. Adding HKE to CPE-pulsed DCs resulted in a significant decrease in T(H)2 cytokine production associated with an increase in IFN-gamma levels and profound attenuation of T-cell proliferation. These effects were linked to HKE-induced TLR-dependent changes in DC reactivity to CPE, especially the production of polarizing cytokines, such as IL-12. TLR signals modulate peanut-induced DC maturation in vitro, leading to changes in the T-cell response to peanut. These TLR effects must be confirmed in vivo and might constitute another alternative for allergen immunotherapies.

Lithium ameliorates phenotypic deficits in a mouse model of fragile X syndrome

As our understanding of the underlying defects in fragile X syndrome (FXS) increases so does the potential for development of treatments aimed at modulating the defects and ameliorating the constellation of symptoms seen in patients. Symptoms of FXS include cognitive disability, hyperactivity, autistic behaviour, seizures and learning deficits. Lithium is a drug used clinically to treat bipolar disorder, and it has been used to treat mood dysregulation in individuals with FXS. We examined whether dietary lithium would alter behavioural and morphological abnormalities in fmr1 knockout (KO) mice. We studied wild-type (WT) and KO mice untreated (control chow) or treated with lithium (0.3% lithium-carbonate-containing chow) commenced at weaning and maintained throughout the experiment. At age 8-12 wk, mice were subjected to the following behavioural tests: open field, social interaction, elevated plus maze, elevated zero maze and passive avoidance. At 13 wk, brains were prepared for Golgi staining and analysis of dendritic spine morphology in medial prefrontal cortex. We found that compared to untreated WT, untreated KO mice were hyperactive and had reduced anxiety, impaired social interactions, and deficits on a learning test. Dendritic spines in medial prefrontal cortex were longer and increased in number. Lithium treatment ameliorated the hyperactivity and reversed impaired social interaction and deficits on the learning test. Lithium treatment also partially normalized general anxiety levels and dendritic spine morphology. Our findings and those from other laboratories on the efficacy of lithium treatment in animal models support further studies in patients with FXS.

Publication bias in reports of animal stroke studies leads to major overstatement of efficacy.

Author SummaryPublication bias is known to be a major problem in the reporting of clinical trials, but its impact in basic research has not previously been quantified. Here we show that publication bias is prevalent in reports of laboratory-based research in animal models of stroke, such that data from as many as one in seven experiments remain unpublished. The result of this bias is that systematic reviews of the published results of interventions in animal models of stroke overstate their efficacy by around one third. Nonpublication of data raises ethical concerns, first because the animals used have not contributed to the sum of human knowledge, and second because participants in clinical trials may be put at unnecessary risk if efficacy in animals has been overstated. It is unlikely that this publication bias in the basic sciences is restricted to the area we have studied, the preclinical modelling of the efficacy of candidate drugs for stroke. A related article in PLoS Medicine (van der Worp et al., doi:10.1371/journal.pmed.1000245) discusses the controversies and possibilities of translating the results of animal experiments into human clinical trials.

Hyperbaric oxygen treatment decreases pain in two nerve injury models

Hyperbaric oxygen (HBO) treatment has been used clinically to treat a variety of ailments, including severe burns and carbon monoxide poisoning, and in research settings has produced promising results when used to treat animal models of inflammatory pain. However, studies examining neuropathic pain or nerve injury models have been limited to physiological assessments and not whether the pain condition improves. The purpose of this study was to evaluate the effect of HBO on two common models of neuropathic pain, L5 ligation and chronic constriction injury (CCI) of the sciatic nerve. Following surgical manipulations, animals demonstrating mechanical hyperalgesia were randomly assigned to either HBO treatment or control for 90 min treatment sessions, after which mechanical sensitivity was assessed at 15 min and 6 h post. Daily HBO sessions, with assessments 15 min post-treatment, continued for two weeks, followed by 5 days of assessment only. The results indicated that both models demonstrated significant improvement in response to treatment over the course of the two-week period, with CCI animals recovering more quickly and maintaining this recovery throughout the post-treatment period. Hyperbaric oxygen treatment appears to be successful in relieving neuropathic pain for an extended period of time, and future research should be aimed at investigating the precise mechanisms underlying this positive effect.

A Comparative Analysis of 7.0-Tesla Magnetic Resonance Imaging and Histology Measurements of Knee Articular Cartilage in a Canine Posterolateral Knee Injury Model

Background There has recently been increased interest in the use of 7.0-T magnetic resonance imaging for evaluating articular cartilage degeneration and quantifying the progression of osteoarthritis. Purpose The purpose of this study was to evaluate articular cartilage cross-sectional area and maximum thickness in the medial compartment of intact and destabilized canine knees using 7.0-T magnetic resonance images and compare these results with those obtained from the corresponding histologic sections. Study Design Controlled laboratory study. Methods Five canines had a surgically created unilateral grade III posterolateral knee injury that was followed for 6 months before euthanasia. The opposite, noninjured knee was used as a control. At necropsy, 3-dimensional gradient echo images of the medial tibial plateau of both knees were obtained using a 7.0-T magnetic resonance imaging scanner. Articular cartilage area and maximum thickness in this site were digitally measured on the magnetic resonance images. The proximal tibias were processed for routine histologic analysis with hematoxylin and eosin staining. Articular cartilage area and maximum thickness were measured in histologic sections corresponding to the sites of the magnetic resonance slices. Results The magnetic resonance imaging results revealed an increase in articular cartilage area and maximum thickness in surgical knees compared with control knees in all specimens; these changes were significant for both parameters (P <.05 for area; P<.01 for thickness). The average increase in area was 14.8% and the average increase in maximum thickness was 15.1%. The histologic results revealed an average increase in area of 27.4% (P <.05) and an average increase in maximum thickness of 33.0% (P 5 .06). Correlation analysis between the magnetic resonance imaging and histology data revealed that the area values were significantly correlated (P <.01), but the values for thickness obtained from magnetic resonance imaging were not significantly different from the histology sections (P > 1). Conclusion These results demonstrate that 7.0-T magnetic resonance imaging provides an alternative method to histology to evaluate early osteoarthritic changes in articular cartilage in a canine model by detecting increases in articular cartilage area. Clinical Relevance The noninvasive nature of 7.0-T magnetic resonance imaging will allow for in vivo monitoring of osteoarthritis progression and intervention in animal models and humans for osteoarthritis.

Experimental Therapies in Hypertrophic Cardiomyopathy

The quintessential clinical diagnostic phenotype of human hypertrophic cardiomyopathy (HCM) is primary cardiac hypertrophy. Cardiac hypertrophy is also a major determinant of mortality and morbidity including the risk of sudden cardiac death (SCD) in patients with HCM. Reversal and attenuation of cardiac hypertrophy and its accompanying fibrosis is expected to improve morbidity as well as decrease the risk of SCD in patients with HCM.The conventionally used pharmacological agents in treatment of patients with HCM have not been shown to reverse or attenuate established cardiac hypertrophy and fibrosis. An effective treatment of HCM has to target the molecular mechanisms that are involved in the pathogenesis of the phenotype. Mechanistic studies suggest that cardiac hypertrophy in HCM is secondary to activation of various hypertrophic signaling molecules and, hence, is potentially reversible. The hypothesis is supported by the results of genetic and pharmacological interventions in animal models. The results have shown potential beneficial effects of angiotensin II receptor blocker losartan, mineralocorticoid receptor blocker spironolactone, 3-hydroxy-3-methyglutaryl-coenzyme A reductase inhibitors simvastatin and atorvastatin, and most recently, N-acetylcysteine (NAC) on reversal or prevention of hypertrophy and fibrosis in HCM. The most promising results have been obtained with NAC, which through multiple thiol-responsive mechanisms completely reversed established cardiac hypertrophy and fibrosis in three independent studies. Pilot studies with losartan and statins in humans have established the feasibility of such studies. The results in animal models have firmly established the reversibility of established cardiac hypertrophy and fibrosis in HCM and have set the stage for advancing the findings in the animal models to human patients with HCM through conducting large-scale efficacy studies.

Sugar‐free Antibodies—The Bacterial Solution to Autoimmunity?

The enzyme EndoS from Streptococcus pyogenes is an immunomodulatory molecule hydrolyzing the conserved glycans in the effector part of immunoglobulin G (IgG). EndoS is remarkably specific for IgG, and hydrolysis has profound effects on IgG effector functions. EndoS pretreatment of IgG, or direct administration to animals with experimental antibody-mediated autoimmune diseases, inhibits development of disease or cures animals from established disease. The properties of EndoS make it a unique experimental tool and an attractive alternative to current therapies of conditions involving pathogenic antibodies. This review describes the discovery of EndoS, the effects of EndoS on IgG effector functions in vitro and in vivo, the biotechnological potential of EndoS, and the outcomes of EndoS treatment in animal models of autoimmunity.

High dose mistletoe treatment inhibits tumor growth and improves survival of B16.F10 melanoma bearing mice

Melanoma is the most aggressive skin cancer with increasing incidence and still poor prognosis. Because of the low response rates (˜20%) towards the standard chemotherapeutic agent dacarbazine (DTIC), new approaches for melanoma therapy are required. Our therapeutical approach is the use of mistletoe extractions for cancer and especially melanoma treatment in animal models. We show here antitumoral effects of aqueous mistletoe extracts in a mouse melanoma model with immunocompetent C57BL/6 mice and syngeneic B16.F10 melanoma cells.

Antidepressants induce regionally discrete, sex-dependent changes in brain's glutamate content

Growing evidence suggests the involvement of glutamate in mood disorders and in the response to antidepressants. However, there is no information regarding a hypothesized sex-dependent glutamatergic modulation following treatment in animal models of depression. We comparatively assayed in male and female Flinders and control Sprague–Dawley rats glutamate and aspartate tissue levels in the prefrontal cortex, hippocampus and nucleus accumbens following 14-day treatment with either 10 mg/kg clomipramine or mirtazapine, intraperitoneally. Clomipramine increased cortical glutamate in both sexes and hippocampal glutamate only in female Flinders rodents. Mirtazapine had no effect on cortical glutamate content but increased hippocampal glutamate in both Flinders sexes. Neither mirtazapine nor clomipramine altered glutamate levels in the nucleus accumbens. There were no any significant differences in aspartate levels. However, in control male SD rats clomipramine and mirtazapine significantly decreased cortical aspartate levels. Our results indicate that two different types of established antidepressants induce a brain region-specific effect on glutamate content. This effect is also characterized by sex-dependent differences mainly in the hippocampus, highlighting a differentiated response of glutamate to distinct antidepressants.

Gender differences on MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in C57BL/6 mice

The aim of this study was to investigate the impact of gender difference in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animal model of Parkinson's disease (PD). In the present study, we investigated the time-dependent alterations of dopamine and its metabolites, striatal tyrosine hydroxylase (TH) protein, dopamine transporter (DAT) protein, glial fibrillary acidic protein (GFAP) protein and midbrain TH protein and motor function in male and female mice 5 h and 1, 3 and 7 days after four administrations of MPTP (20 mg/kg) at 2-h intervals. The present study showed that the decrease of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) content in female mice was more pronounced than that in male animals 1, 3 and 7 days after MPTP treatment. Our Western blot analysis study also demonstrated that the decrease of both striatal and midbrain TH protein levels in female mice was more pronounced than that in male animals from 1 to 7 days after MPTP treatment. As compared to male mice, in contrast, the increase of striatal GFAP protein levels in female mice was observed from 5 h to 7 days after MPTP treatment. Furthermore, the present study showed that motor deficits were found in both male and female mice 1 and 7 days after MPTP treatment. In the present study, moreover, the decrease of striatal DAT protein levels in female mice was more pronounced than that in male animals 1, 3 and 7 days after MPTP treatment. These results demonstrate that our administrations of MPTP at 2-h intervals can cause more severe damage in female mice as compared with male animals. The gender difference may be due to the decrease of DAT expression caused by MPTP. Thus our findings provide further valuable information for the pathogenesis of PD.

The Bcl-2 Family Antagonist ABT-737 Significantly Inhibits Multiple Animal Models of Autoimmunity

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.

Thioredoxin Suppresses the Contact Hypersensitivity Response by Inhibiting Leukocyte Recruitment During the Elicitation Phase

Thioredoxin, a redox-regulating protein that scavenges reactive oxygen species, appears to show an excellent antiinflammatory effect in treating animal models of various human inflammatory diseases. The aim of this study was to clarify whether thioredoxin is useful for treating inflammatory skin diseases, such as contact dermatitis, caused by epicutaneous exposure to environmental and occupational antigens. The allergic contact hypersensitivity response was suppressed in thioredoxin-transgenic mice. This suppressive effect of thioredoxin appeared to be via the inhibition of the efferent limb of contact hypersensitivity because administration of recombinant thioredoxin suppressed the inflammatory response in the elicitation phase but not in the induction phase. Adoptive-transfer studies revealed that the host environment, but not donor leukocytes, is critical in this suppressive effect. In thioredoxin-transgenic mice, the infiltration of neutrophils in the elicitation site was diminished, whereas the migratory function of cutaneous dendritic cells and hapten-specific cell proliferation were not disturbed. Thioredoxin-transgenic mice had also an attenuated inflammatory response to croton oil. These findings suggest that thioredoxin prevents skin inflammatory responses and could be a suitable candidate for the treatment of contact dermatitis.

Validation of cerebral blood perfusion imaging as a modality for quantitative pharmacological MRI in rats

The aim of this study was to validate continuous arterial spin labeling (CASL) as a quantitative imaging modality for pharmacological MRI (phMRI) based on local cerebral blood perfusion. Specifically, the capability of CASL to assess brain-activity signatures of pharmacological interventions in animal models was evaluated with respect to drug discovery in diseases of the central nervous system (CNS). Perfusion as a surrogate for neuronal activity was measured in various brain areas of the rat. The validation approach was threefold. First, perfusion was shown to reliably reflect differential effects of anesthesia on striatal activation. Different baseline levels and different temporal response profiles after amphetamine challenges under isoflurane, propofol, ketamine, and alpha-chloralose anesthesia were consistent with known properties of these anesthetics. Second, remarkable consistency of multi-area baseline perfusion patterns between independent groups of animals confirmed the notion that CASL is highly reproducible and thus particularly suitable for long-term longitudinal studies. Third, administration of the well-characterized psychotomimetic compounds amphetamine and phencyclidine (PCP) elicited dose-dependent activation patterns that were related to the drugs' particular interactions with the dopaminergic and glutamatergic systems, respectively. In conclusion, perfusion-based phMRI is a robust, reliable and valid quantitative technique suitable for evaluating brain-activation patterns in animal models of CNS diseases.

Neuroinflammation and Neuroprotective Strategies in Acute Ischaemic Stroke - from bench to bedside

Cerebrovascular disease is one of the commonest causes of disability and mortality worldwide. Over the past two decades, a tremendous amount of research has been undertaken into developing effective therapeutic strategies for the treatment of acute stroke. Unfortunately, many neuroprotective agents that have shown successful results in treating animal models of acute stroke have failed to translate into clinical treatments. Only tissue-plasminogen activator (t-PA) is currently licensed for use in the treatment of acute ischaemic stroke. One of the important pathophysiological mechanisms involved during the acute phase of stroke is neuroinflammation. This review article will discuss the molecular aspects of neuroinflammation in acute ischaemic stroke and potential therapeutic strategies as part of translational medicine research.

Overcoming biological barriers to in vivo efficacy of antisense oligonucleotides

Antisense oligonucleotides as a therapeutic platform have been slow to progress since the approval of the first antisense drug in 1998. Recently, there have been several examples of convincing antisense interventions in animal models and promising clinical trial data. This review considers the factors determining the success of antisense oligonucleotides as therapeutic agents. In order to produce target knockdown after systemic delivery, antisense oligonucleotides must avoid nuclease degradation, reticuloendothelial-system uptake and rapid renal excretion, and extravasate to the target cell type outside the vasculature. They then must enter the target cell, and escape the endosome-lysosome pathway so as to be free to interact with the target mRNA. We consider the significance of these limiting factors based on the literature and our own experience using systemic administration of antisense oligonucleotides.

Personalised Genetic Intervention for Duchenne Muscular Dystrophy: Antisense Oligomers and Exon Skipping

Duchenne muscular dystrophy (DMD) arises from protein-truncating mutations in the large dystrophin gene that preclude synthesis of a functional protein that primarily stabilizes muscle fibre membranes. The absence of dystrophin leads to this most common and serious form of childhood muscle-wasting. Since the identification of the dystrophin gene in 1987, cell and gene repair or replacement therapies have been evaluated for DMD treatment and one genetic intervention, exon skipping, is now in clinical trials. Antisense oligomers have been designed to redirect dystrophin splicing patterns so that targeted exons may be removed from a defective dystrophin pre-mRNA to either restore the reading frame of a deletion, or excise an in-frame exon corrupted by a nonsense mutation or microinsertion/ deletion. This review discusses the evolution of oligomer induced exon skipping, including in vitro applications, evaluation of different oligomer chemistries, the treatment of animal models and alternative exon skipping strategies involving viral expression cassettes and ex vivo manipulation of stem cells. The discussion culminates with the current clinical trials and the great challenges that lie ahead. The major obstacle to the implementation of personalised genetic treatments to address the many different mutations that can lead to DMD, are considered to be establishing effective treatments for the different patients and their mutations. Furthermore, the view of regulatory authorities in assessing preclinical data on potentially scores of different but class-specific compounds will be of paramount importance in expediting the clinical application of exon skipping therapy for this serious and relentlessly progressive muscle wasting disease. Keywords: Antisense oligonucleotides, exon skipping, duchenne muscular dystrophy, morpholino, clinical trials, dystrophin, personalized medicine, pre-mRNA splicing

Personalised Genetic Intervention for Duchenne Muscular Dystrophy: Antisense Oligomers and Exon Skipping

Duchenne muscular dystrophy (DMD) arises from protein-truncating mutations in the large dystrophin gene that preclude synthesis of a functional protein that primarily stabilizes muscle fibre membranes. The absence of dystrophin leads to this most common and serious form of childhood muscle-wasting. Since the identification of the dystrophin gene in 1987, cell and gene repair or replacement therapies have been evaluated for DMD treatment and one genetic intervention, exon skipping, is now in clinical trials. Antisense oligomers have been designed to redirect dystrophin splicing patterns so that targeted exons may be removed from a defective dystrophin pre-mRNA to either restore the reading frame of a deletion, or excise an in-frame exon corrupted by a nonsense mutation or micro-insertion/deletion. This review discusses the evolution of oligomer induced exon skipping, including in vitro applications, evaluation of different oligomer chemistries, the treatment of animal models and alternative exon skipping strategies involving viral expression cassettes and ex vivo manipulation of stem cells. The discussion culminates with the current clinical trials and the great challenges that lie ahead. The major obstacle to the implementation of personalised genetic treatments to address the many different mutations that can lead to DMD, are considered to be establishing effective treatments for the different patients and their mutations. Furthermore, the view of regulatory authorities in assessing preclinical data on potentially scores of different but class-specific compounds will be of paramount importance in expediting the clinical application of exon skipping therapy for this serious and relentlessly progressive muscle wasting disease.