Rebecca J. Fairclough

Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches

Duchenne muscular dystrophy (DMD) is a devastating progressive disease for which there is currently no effective treatment except palliative therapy. There are several promising genetic approaches, including viral delivery of the missing dystrophin gene, read-through of translation stop codons, exon skipping to restore the reading frame and increased expression of the compensatory utrophin gene. The lessons learned from these approaches will be applicable to many other disorders.

Daily Treatment with SMTC1100, a Novel Small Molecule Utrophin Upregulator, Dramatically Reduces the Dystrophic Symptoms in the mdx Mouse

Background Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fibers leading to the gradual depletion of skeletal muscle. There is significant evidence demonstrating that increasing levels of the dystrophin-related protein, utrophin, in mouse models results in sarcolemmal bound utrophin and prevents the muscular dystrophy pathology. The aim of this work was to develop a small molecule which increases the levels of utrophin in muscle and thus has therapeutic potential. Methodology and Principal Findings We describe the in vivo activity of SMT C1100; the first orally bioavailable small molecule utrophin upregulator. Once-a-day daily-dosing with SMT C1100 reduces a number of the pathological effects of dystrophin deficiency. Treatment results in reduced pathology, better muscle physiology leading to an increase in overall strength, and an ability to resist fatigue after forced exercise; a surrogate for the six minute walk test currently recommended as the pivotal outcome measure in human trials for DMD. Conclusions and Significance This study demonstrates proof-of-principle for the use of in vitro screening methods in allowing identification of pharmacological agents for utrophin transcriptional upregulation. The best compound identified, SMT C1100, demonstrated significant disease modifying effects in DMD models. Our data warrant the full evaluation of this compound in clinical trials in DMD patients.

Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.

Progress in therapy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a devastating muscular dystrophy of childhood. Mutations in the dystrophin gene destroy the link between the internal muscle filaments and the extracellular matrix, resulting in severe muscle weakness and progressive muscle wasting. There is currently no cure and, whilst palliative treatment has improved, affected boys are normally confined to a wheelchair by 12 years of age and die from respiratory or cardiac complications in their twenties or thirties. Therapies currently being developed include mutation‐specific treatments, DNA‐ and cell‐based therapies, and drugs which aim to modulate cellular pathways or gene expression. This review aims to provide an overview of the different therapeutic approaches aimed at reconstructing the dystrophin‐associated protein complex, including restoration of dystrophin expression and upregulation of the functional homologue, utrophin.

Pharmacologically targeting the primary defect and downstream pathology in Duchenne muscular dystrophy.

DMD is a devastatingly progressive muscle wasting disorder of childhood that significantly shortens life expectancy. Despite efforts to develop an effective therapy that dates back over a century, clinical interventions are still restricted to management of symptoms rather than a cure. The rationale to develop effective therapies changed in 1986 with the discovery of the dystrophin gene. Since then extensive research into both the molecular basis and pathophysiology of DMD has paved the way not only for development of strategies which aim to correct the primary defect, but also towards the identification of countless therapeutic targets with the potential to alleviate the downstream pathology. In addition to gene and cell-based therapies, which aim to deliver the missing gene and/or protein, an exciting spectrum of pharmacological approaches aimed at modulating therapeutic targets within DMD muscle cells through the use of small drugs are also being developed. This review presents promising pharmacological approaches aimed at targeting the primary defect, including suppression of nonsense mutations and functional compensation by upregulation of the dystrophin homologue, utrophin. Downstream of the primary membrane fragility, inflammation and fibrosis are reduced by blocking NF-κB, TGF-α and TGF-β, and free radical damage has been targeted using antioxidants and dietary/nutritional supplements. There are new hopes that ACE and PDE5 inhibitors can protect against skeletal as well as cardiac pathology, and modulating Ca2+ influx, NO, BMP, protein degradation and the mitochondrial permeability pore hold further promise in tackling the complex pathogenesis of this multifaceted disorder.

P.13.14 Future clinical and biomarker development for SMTC1100, the first utrophin modulator to enter clinical trials for Duchenne Muscular Dystrophy (DMD)

Utrophin modulation i.e. the re-programming of utrophin transcription such that utrophin RNA and protein is continually expressed in mature fibres is expected to be disease modifying in all genetic forms of DMD. SMTC1100 is a small molecule utrophin modulator demonstrating significant benefit on the muscular dystrophy in the dystrophin deficient mdx mouse. These data led to the nomination of SMTC1100 as the candidate for evaluation in DMD clinical trials. In 2012 we reported that SMTC1100 successfully completed a Phase 1 healthy volunteer trial in which an oral paediatric formulation was deemed safe and well tolerated with plasma levels well above those determined to be efficacious in cells and animals. Plans for the first patient trials of SMTC1100 have been developed consisting of two components; a safety and dose finding study in DMD boys in late 2013 followed by a proof of concept study in 2014. In order for proof of concept to be demonstrated in patients, a multicomponent biomarker strategy has been implemented that comprises of two modules; the increase of utrophin levels and evidence of reduction in muscle regeneration. To demonstrate increased utrophin derived from drug treatment above that normally found in regenerating DMD muscle, we aim to quantify utrophin RNA, total utrophin protein and utrophin fibre localisation derived from pre- and post-dose biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of newly regenerating fibres as determined by the presence of neonatal and foetal myosin will be calculated from the biopsies. Using serum and urine samples we will quantify the levels of specific miRNAs associated with fibre leakage and peptide markers of active fibrosis which characterises fibre damage and degeneration respectively. We will present the patient clinical trial plans and data from candidate biomarkers tested both in DMD samples and dystrophin deficient animals.

G.P.89

DMD is a devastating X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches (e.g. exon skipping, read through of stop codons, gene therapy) are being developed. By pharmacologically modulating the dystrophin-related protein utrophin, we aim to develop a therapy applicable to all DMD patients by targeting the primary defect and restoring sarcolemmal stability. In partnership with Summit plc we previously developed SMT C1100; a small molecule utrophin modulator that reduced dystrophic symptoms in the mdx mouse. As a potential first-in-class molecule, SMT C1100 recently successfully completed a Phase 1a trial. DMD patients are currently being dosed in an ongoing Phase 1b trial for which a multicomponent biomarker strategy has been implemented with a quantification of utrophin level, regeneration, fibrosis, inflammation and analysis of specific miRNAs. The successful clinical progression to date provides crucial proof-of-concept for the strategy we developed. We are now optimising next generation utrophin modulator molecule which were discovered using an improved drug screening assay based on immortalised myoblasts from the utrophin luciferase knock-in mouse. This enables us to screen utrophin in its genomic context, better mimicking the in vivo situation and enabling identification of compounds which modulate utrophin through regulatory pathways outside of the 8.4kb promoter fragment used in our previous screen. Thus, we identified multiple structural classes which significantly modulate utrophin expression in both mouse and human DMD myoblasts. New compounds exhibit favourable solubility, stability, oral absorption and are well tolerated in the mouse and structure–activity studies are underway, with the objective to improve compound effectiveness. The two of the best new utrophin modulators are currently being studied in pre-clinical in vivo studies in mdx mice.

Utrophin upregulation in DMD therapy: current status and new tools for the future

P04 Correction of FKRP function via RNA trans-splicing S. Farmer, S. Lorain, A. Thrasher, L. Garcia, F. Muntoni, F. Conti. Dubowitz Neuromuscular Centre, UCL Institute of Child Health, 30 Guildford Street, London WC1N 1EH, UK; Institut National de Sante et de Recherche Medicale, Groupe Myologie, Universite Pierre et Marie Curie, Paris, France; Molecular Immunology Unit, UCL Institute of Child Health, 30 Guildford Street, London WC1N 3BG, UK