Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: Implication for clinical trials

Duchenne muscular dystrophy (DMD) is characterised by the absence of dystrophin in muscle biopsies, although residual dystrophin can be present, either as dystrophin-positive (revertant) fibres or traces. As restoration of dystrophin expression is the end point of clinical trials, such residual dystrophin is a key factor in recruitment of patients and may also confound the analysis of dystrophin restoration in treated patients, if, as previously observed in the mdx mouse, revertant fibres increase with age. In 62% of the diagnostic biopsies reports of 65 DMD patients studied, traces or revertants were recorded with no correlation between traces or revertants, the patients' performance, or corticosteroids response. In nine of these patients, there was no increase in traces or revertants in biopsies taken a mean of 8.23 years (5.8-10.4 years) after the original diagnostic biopsy. This information should help in the design and execution of clinical trials focused on dystrophin restoration strategies. (C) 2010 Elsevier B.V. All rights reserved

Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms

A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

BACKGROUND Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". METHODOLOGY/PRINCIPAL FINDINGS Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. CONCLUSIONS/SIGNIFICANCE Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD

Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices

Neuronal activity in the respiratory network is functionally dependent on inhibitory synaptic transmission. Using two-photon excitation microscopy, we analyzed the integration of glycinergic neurons in the isolated inspiratory pre-Bötzinger complex-driven network of the rhythmic slice preparation. Inspiratory (96%) and ‘tonic’ expiratory neurons (4%) were identified via an increase or decrease, respectively, of the cytosolic free calcium concentration during the inspiratory-related respiratory burst. Furthermore, in BAC-transgenic mice expressing EGFP under the control of the GlyT2-promoter, 50% of calcium-imaged inspiratory neurons were glycinergic. Inspiratory bursting of glycinergic neurons in the slice was confirmed by whole-cell recording. We also found glycinergic neurons that receive phasic inhibition from other glycinergic neurons. Our calcium imaging data show that glycinergic neurons comprise a large population of inspiratory neurons in the pre-Bötzinger complex-driven network of the rhythmic slice preparation

Stakeholder cooperation to overcome challenges in orphan medicine development: The example of Duchenne muscular dystrophy

Duchenne muscular dystrophy is a rare, progressive, muscle-wasting disease leading to severe disability and premature death. Treatment is currently symptomatic, but several experimental therapies are in development. Implemented care standards, validated outcome measures correlating with clinical benefit, and comprehensive information about the natural history of the disease are essential for regulatory approval of any treatment. However, for Duchenne muscular dystrophy and other rare diseases, these requirements are not always in place when potential therapies enter the clinical trial phase. A cooperative effort of stakeholders in Duchenne muscular dystrophy-including representatives from patients' groups, academia, industry, and regulatory agencies-is aimed at addressing this shortfall by identifying strategies to overcome challenges, developing the tools needed, and collecting relevant data. An open and constructive dialogue among European stakeholders has positively affected development of treatments for Duchenne muscular dystrophy; this approach could serve as a paradigm for development of treatments for rare diseases in general

Delivery of oligonucleotide‐based therapeutics: challenges and opportunities

Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics

Report of a TREAT-NMD/World Duchenne Organisation Meeting on Dystrophin Quantification Methodology

Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, outline the gaps with regards to validating the methods for robust clinical applications prior to regulatory agency review, and to align future efforts towards further optimizing these methods. The workshop facilitated a constructive but also critical discussion on the potential and limitations of techniques currently used in the field of translational research (western blot and immunofluorescence analysis) and emerging techniques (mass spectrometry and capillary western immunoassay). Notably, all participants reported variation in dystrophin levels between muscle biopsies from different healthy individuals and agreed on the need for a common reference sample

Activation of the SPHK/S1P signalling pathway is coupled to muscarinic receptor-dependent regulation of peripheral airways

BACKGROUND: In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M(2)R and M(3)R). Cholinergic hypersensitivity is associated with chronic obstructive pulmonary disease and asthma, and therefore the identification of muscarinic signaling pathways are of great therapeutic interest. A pathway that has been shown to be activated via MR and to increase [Ca(2+)](i )includes the activation of sphingosine kinases (SPHK) and the generation of the bioactive sphingolipid sphingosine 1-phosphate (S1P). Whether the SPHK/S1P signaling pathway is integrated in the muscarinic control of peripheral airways is not known. METHODS: To address this issue, we studied precision cut lung slices derived from FVB and M(2)R-KO and M(3)R-KO mice. RESULTS: In peripheral airways of FVB, wild-type, and MR-deficient mice, SPHK1 was mainly localized to smooth muscle. Muscarine induced a constriction in all investigated mouse strains which was reduced by inhibition of SPHK using D, L-threo-dihydrosphingosine (DHS) and N, N-dimethyl-sphingosine (DMS) but not by N-acetylsphingosine (N-AcS), a structurally related agent that does not affect SPHK function. The initial phase of constriction was nearly absent in peripheral airways of M(3)R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M(2)R-KO mice. Quantitative RT-PCR revealed that the disruption of the M(2)R and M(3)R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways. CONCLUSION: These results demonstrate that the SPHK/S1P signaling pathway contributes to cholinergic constriction of murine peripheral airways. In addition, our data strongly suggest that SPHK is activated via the M(2)R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance