A Potter

Utrophin-Dystrophin-Deficient Mice as a Model for Duchenne Muscular Dystrophy

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.

Genomic Variation and Gene Conversion in Spinal Muscular Atrophy: Implications for Disease Process and Clinical Phenotype

Autosomal recessive spinal muscular atrophy (SMA) is classified, on the basis of age at onset and severity, into three types: type I, severe; type II, intermediate; and type III, mild. The critical region in 5q13 contains an inverted repeat harboring several genes, including the survival motor neuron (SMN) gene, the neuronal apoptosis inhibitory protein (NAIP) gene, and the p44 gene, which encodes a transcription-factor subunit. Deletion of NAIP and p44 is observed more often in severe SMA, but there is no evidence that these genes play a role in the pathology of the disease. In > 90% of all SMA patients, exons 7 and 8 of the telomeric SMN gene (SMNtel) are not detectable, and this is also observed in some normal siblings and parents. Point mutations and gene conversions in SMNtel suggest that it plays a major role in the disease. To define a correlation between genotype and phenotype, we mapped deletions, using pulsed-field gel electrophoresis. Surprisingly, our data show that mutations in SMA types II and III, previously classed as deletions, are in fact due to gene-conversion events in which SMNtel is replaced by its centromeric counterpart, SMNcen. This results in a greater number of SMNcen copies in type II and type III patients compared with type I patients and enables a genotype/phenotype correlation to be made. We also demonstrate individual DNA-content variations of several hundred kilobases, even in a relatively isolated population from Finland. This explains why no consensus map of this region has been produced. This DNA variation may be due to a midisatellite repeat array, which would promote the observed high deletion and gene-conversion rate.

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.

Rescue of skeletal muscle alpha-actin-null mice by cardiac (fetal) alpha-actin.

Skeletal muscle α-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac α-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.

Discovery of 2-arylbenzoxazoles as upregulators of utrophin production for the treatment of Duchenne muscular dystrophy.

A series of novel 2-arylbenzoxazoles that upregulate the production of utrophin in murine H2K cells, as assessed using a luciferase reporter linked assay, have been identified. This compound class appears to hold considerable promise as a potential treatment for Duchenne muscular dystrophy. Following the delineation of structure-activity relationships in the series, a number of potent upregulators were identified, and preliminary ADME evaluation is described. These studies have resulted in the identification of 1, a compound that has been progressed to clinical trials.

Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.

Expression profiling in spinal muscular atrophy reveals an RNA binding protein deficit

Spinal muscular atrophy is a common neuromuscular disorder caused by deletions or mutations within the survival motor neuron gene. The reason for specific motor neuron loss within the disease is still unclear. Expression profiling has been carried out in two models of spinal muscular atrophy; the heterozygote mouse model and human primary muscle cultures from a spinal muscular atrophy patient. A group of RNA binding proteins are up-regulated in spinal muscular atrophy motor neurons. One such protein, BRUNOL3, is highly expressed within spinal cord and muscle and also at the same developmental stage as survival motor neuron. The differential expression of Brunol3 has been confirmed with real-time RT-PCR in spinal cord and muscle of three different models of spinal muscular atrophy. BRUNOL3 has been shown to co-localise with survival motor neuron in the nuclei of neuronal cells and to co-immunoprecipitate with Smn in mouse brain. This is the first time that a link has been established between RNA binding proteins and survival motor neuron within motor neurons.

Dystrophin and utrophin do not play crucial roles in nonmuscle tissues in mice

The loss of full‐length dystrophin from skeletal muscle leads to the clinical features of Duchenne muscular dystrophy. Both Dp71, a C‐terminal dystrophin isoform, and the dystrophin‐related protein, utrophin, are present at high levels in many nonmuscle tissues. To investigate the roles of these proteins in nonmuscle tissues, mice were generated null for utrophin, and deficient in all dystrophin isoforms. These mice reach adulthood and do not appear to have any devastating pathology in nonmuscle tissues.

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