The use of splice‐switching antisense therapy is highly promising, with a wealth of pre‐clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders ...has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery. We are a group of researchers closely involved in the development of these therapies and would like to communicate our discussions concerning the validity of standard methodologies currently used in their pre‐clinical development, the gaps in current knowledge and the pertinent challenges facing the field. We therefore make recommendations in order to focus future research efforts and facilitate a wider application of therapeutic antisense oligonucleotides.
The use of splice‐switching antisense therapy is promising; nevertheless, its potential to treat a variety of disorders has yet to be realized. This review discusses successes and obstacles of this approach and provides a forward look into wider application of therapeutic antisense oligonucleotides.
Central Core Disease is a myopathy resulting generally from a mutation in the RYR1 gene, encoding the skeletal muscle calcium release channel RyR1. No treatment is currently available for this ...disease. We studied a pathological situation in which an affected child harbors two recessive mutations, resulting in a massive reduction in the RyR1 amount. The paternal mutation inducing the inclusion of a new in frame exon in the mRNA of RyR1, resulted in the insertion of additional amino-acids and destabilization of the protein. We hypothesized that inducing the skipping of this exon would be sufficient to restore RyR1 expression and normalization of calcium releases. We developed U7-AON lentiviral vectors to induce exon-skipping on affected primary muscle cells. The efficiency of the exon skipping at the mRNA level, at the protein level and at the functional level using calcium imaging were evaluated. We observed in these affected primary muscle culture a reduction in the inclusion of the additional exon, an increase in the RyR1 protein expression, and a restoration of normal calcium releases. This study is the first demonstration of the potential of exon skipping for the therapy of Central Core Disease, from the molecular to the functional level.
The most promising gene therapy approaches to treat Duchenne Muscular Dystrophy (DMD) is currently the use of the exon skipping strategy as shown by clinical trials underway. However, truncated ...dystrophins resulting from exon skipping may be more or less efficacious depending on the effect of the truncation on the protein function. In particular, the neuronal nitric oxide synthase (nNOS) binding site to the dystrophin has been defined on the repeats 16 and 17 (R16/17). Considering that R16/17 are encoded by exons 42–45 of dystrophin mRNA, an alteration in this zone could impact on the integrity of the nNOS binding site preventing the proper binding of nNOS to dystrophin. Several studies have provided convincing evidence that mis-localization of nNOS and cytosolic NO impair skeletal muscle contractibility via nitrosylative modification of the sarcoplasmic reticulum calcium-release-channel RyR1. These data point towards nitrosylation-induced leakiness of the RyR1 as a molecular mechanism underlying muscle weakness and highlight that the dystrophin rescue has to take into account its functional properties such as nNOS binding. Considering these facts, we have evaluated the level of dystrophin restoration needed to recover, qualitatively and quantitatively, a correct location of nNOS. For this study, we have collected biopsies from GRMD dogs (the DMD model) treated by exon skipping strategy and for whom muscles display a range from 5% to 95% of dystrophin positive fibers. The location and the activity of nNOS as well as the status of RyR1 nitrosylation have been investigated following the rank of dystrophin rescue. This study allowed to define the level of dystrophin restoration necessary for the proper nNOS localization and for the return to the normalization of the RyR1 status, two crucial parameters to be investigated in DMD therapy for which both, the dystrophin restoration and the muscular function rescue, are the goal.
Adeno Associated virus serotype 8 (AAV8) is of particular interest as a vector for pre-clinical and clinical trial for Duchenne Muscular Dystrophy (DMD). In several cell lines, this vector has been ...shown to enter cells through clathrin-mediated endocytosis followed by a trafficking through the microtubule network in various endosomal compartments toward the nucleus. To efficiently transduce cells, AAV must undergo multiple levels of regulation in these cellular compartments. In DMD, dystrophin deficiency results in disturbed balance of cellular events i.e., fiber centronucleation, disorganized cytoskeleton, presence of fibrosis. We have recently described a loss of virion genomes from both dogs and mice models of DMD treated with therapeutic molecules vectorized in AAV. Indeed, the pathophysiological state of DMD muscle should impact on virions fate and subsequently affect crucial steps for AAV effectiveness as viral uncoating, viral genome maintenance and consequently, the transduction efficiency of AAV. Our project aims to characterize cellular uptake and intracellular transport of AAV8 in DMD muscular cells, with the goal of optimizing AAV vector use to get the best transduction efficiency with the lowest AAV dose. Our first data showed that AAV8-GFP was less efficient to transduce DMD and control primary muscular cells compared to HeLa cells. Moreover, AAV8 traffics through same endosomal compartment in DMD and control myoblasts, but at different rates during early time points of the transduction. These results suggest that in muscle cells, AAV8 uses different entry and trafficking pathways from those previously described in HeLa cells and that dystrophic cellular status could affect subcellular processing of the vector particles. We will specify the relationship between AAV8 vector entry, trafficking, uncoating, and transduction efficiency in vitro in primary myoblasts/myotubes of DMD patients and controls.
Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy-dependent muscle function remains largely unexplored. This ...question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here, we show in mice, that 4-month pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling downregulates porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phophorus Magnetic Resonance Spectroscopy (31P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.
Abstract Duchenne (DMD) and Becker (BMD) muscular dystrophies are muscle-wasting diseases caused by mutations in the DMD gene encoding dystrophin. Usually, out-of-frame deletions give rise to DMD ...whereas in-frame deletions result in BMD. BMD patients exhibit a less severe disease because an abnormal but functional dystrophin is produced. This is the rationale for attempts to correct the reading frame by using an exon-skipping strategy. In order to apply this approach to a larger number of patients, a strategy of exons 45–55 multi-skipping has been proposed, because it would correct the mRNA reading frame in almost 75% of DMD patients with a deletion. The resulting dystrophin lacks part of the binding site for the neuronal nitric oxide synthase (nNOSμ), which normally binds to spectrin-like repeats 16 and 17 of dystrophin. Knowing that these domains are encoded by exons 42–45, we investigated the nNOSμ status in muscle biopsies from twelve BMD patients with deletions of exons 45–55. We found a wide spectrum of nNOSμ expression and localization. The strictly cytosolic mislocalization of nNOSμμ was associated with the more severe phenotypes. The cytosolic NO production correlated with both hypernitrosylation of the sarcoplasmic reticulum calcium-release-channel RyR1 and release of calstabin-1, a central hub to Ca2+ signaling and contraction in muscle. This study shows that the terminal truncation of the nNOS binding domain in the “therapeutic” del45–55 dystrophin is not innocuous since, in some cases, it may perturb the nNOS dependent stability of the RyR1/calstabin-1 complex.
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