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Spinal muscular atrophy (SMA) is an autosomal recessive disease causing degeneration of lower motor neurons and muscle atrophy. One therapeutic avenue for SMA is targeting signaling pathways in muscle to ameliorate atrophy. Muscle Atrophy F-box, MAFbx, and Muscle RING Finger 1, MuRF1, are muscle-specific ubiquitin ligases upregulated in skeletal and cardiac muscle during atrophy. Homozygous knock-out of MAFbx or MuRF1 causes muscle sparing in adult mice subjected to atrophy by denervation. We wished to determine whether blockage of the major muscle atrophy pathways by deletion of MAFbx or MuRF1 in a mouse model of SMA would improve the phenotype. Deletion of MAFbx in the Δ7 SMA mouse model had no effect on the weight and the survival of the mice while deletion of MuRF1 was deleterious. MAFbx(-/-)-SMA mice showed a significant alteration in fiber size distribution tending towards larger fibers. In skeletal and cardiac tissue MAFbx and MuRF1 transcripts were upregulated whereas MuRF2 and MuRF3 levels were unchanged in Δ7 SMA mice. We conclude that deletion of the muscle ubiquitin ligases does not improve the phenotype of a Δ7 SMA mouse. Furthermore, it seems unlikely that the beneficial effect of HDAC inhibitors is mediated through inhibition of MAFbx and MuRF1.

A workshop was held in Zurich (Switzerland) to develop standard procedures and protocols for pre-clinical efficacy studies in mouse models of spinal muscular atrophy (SMA). The workshop was organized by the Network of Excellence TREAT-NMD (Translational Research in Europe – Assessment and Treatment of Neuromuscular Disorders), launched 2007, that addresses the fragmentation currently existing in the evaluation, diagnosis and care of neuromuscular diseases on one side, and in the procedures of drug development from research to the design of clinical trials on the other side. Within this frame, one aspect that needs particular care is the design and performance of pre-clinical animal studies that should predict the efficacy of a new treatment in patients. In the absence of commonly accepted guidelines and standardized protocols, data from different laboratories are often not comparable and the result is a duplication of efforts. The workshop was attended by 30 key researchers from Europe and North America, as well as industry representatives (Santhera Pharmaceuticals, Psychogenics, Genyzme, Trophos) and patient organisations (SMA Foundation, AFM-Association Franc aise contre les Myopathies). The goals of the workshop were (1) to select endpoints that best assess drug efficacy in mouse

Response of biomarkers to treatment in mouse models of spinal muscular atrophy and response of SMA mice to later treatment A. Burghes *, W. Arnold , V. McGovern , S. Duque , J. Li , C. Iyer , K. Corlett , K. von Herrmann , P. Zaworski , K. Chen , S. Kolb , S. Paushkin , D. Kobayashi , S. Rutkove 3 1 Ohio State University, Molecular and Cellular Biochemistry, Columbus, USA; 2 Ohio State University, Neurology, Columbus, USA; 3 Harvard Medical School, Neurology, Boston, USA; 4 SMA Foundation, New York, USA; 5 PharmOptima, Portage, USA; 6 Cydan Development, Cambridge, USA

SMN is expressed in all tissues but its deficiency in SMA selectively affects neurons. Using mice that model severe SMA we have tested whether SMN needs to be replaced in all tissues. Furthermore we have used antisense morpholinos (ASO) directed against ISS-N1 delivered to the nervous system to correct SMA mice. Using mice lines that can either remove mouse SMN expression or restore mouse SMN expression when Cre is expressed we have determined the spatial requirement for SMN in SMA. Removal of SMN from neurons to create SMA neurons in the background of all other cells with normal SMN results in mice with a severe phenotype likewise restoration of SMN to neurons with all other tissues remaining at low levels results in correction. While the Cre drivers clearly indicate that motor neurons are one of the critical neural types for SMN restoration it is not the only neuronal type that requires high SMN levels. Furthermore we have knockdown SMN in pig motor neurons and other neuronal populations in the pig using intrathecal delivery of AAV9-shRNA this resulted in a clear SMA like phenotype. Furthermore with efficient transduction of motor neurons the cell bodies of the motor neuron where retained but ventral root axons showed a marked loss indicating that reduction in SMN results in motor axonal problems prior to the cell body. In mice the intracerebralventricular delivery of ASO has a marked impact on SMA mice we are now investigating the distribution of ASO after intrathecal or ICV delivery initial results indicate it is possible to obtain wide distribution in the nervous system of both adult and neonatal animals.

The dystrophin-glycoprotein complex is considered to be a major trans-sarcolemmal structure which provides a linkage between the subsarcolemmal actin cytoskeleton and the extracellular matrix component laminin. Recently, deficiency of the dystrophin-associated proteins has been shown to play an important role in the molecular pathogenesis of several forms of muscular dystrophy. These include Duchenne muscular dystrophy (DMD), symptomatic DMD carriers, Becker muscular dystrophy and severe childhood autosomal recessive muscular dystrophy with DMD-like phenotype prevalent in North Africa. In Fukuyama-type congenital muscular dystrophy (FCMD), the finding of abnormal expression of the dystrophin-associated proteins may provide a clue to its molecular pathogenesis. These recent findings indicate that the linkage between the subsarcolemmal cytoskeleton and extracellular matrix via the dystrophin-glycoprotein complex is critical for maintaining the integrity of muscle cell function.

Spinal muscular atrophy is caused by loss of SMN1 and retention of SMN2 resulting in low SMN levels. SMN protein functions in assembling snRNP which are critical in splicing of genes. We have developed mice and pig models of SMA. In both mouse and pig we have investigated early and late rescue of SMN. In pig restoration of SMN early via scAAV9-hSMN resulted in marked rescue of phenotype and restoration of Motor Unit Number Estimates (MUNE), Compound Muscle Action Potential (CMAP) and loss of fibrillations. Rescue at time of onset in the pig resulted in improvement of phenotype with MUNE minimally rescued and CMAP showing marked improvement. In SMA Delta 7 mice antisense PMOs where used to restore SMN levels. Restoration of SMN at 4 days had a major impact on survival and restoration of MUNE and CMAP. At 6 days survival was improved with MUNE showing minimal improvement and CMAP markedly improved. At 8 days there was no improvement in any parameter. Thus in both pig and mice early or later induction of SMN improve SMA to different degrees. Furthermore we have laser microdissected motor neurons from both pig and mouse and performed RNAseq to define genes splicing changes in SMA. A limited number of changes specific to SMA motor neurons are detected. To determine which of these are a critical isoform in SMA we are re-introducing the isoform via scAAV9 and testing which alter the phenotype of SMA. In particular we are looking at those that alter the electrophysiological readouts of SMA. Genes that do alter the SMA phenotype represent novel downstream targets that can be viewed as new therapeutic targets.

Spinal muscular atrophy (SMA) is the leading genetic cause of death in infants and results in the loss of motoneurons (MNs). Gene transfer based on systemic delivery of scAAV9 viral vector expressing human SMN (hSMN) has successfully rescued severe SMA mice and lead to the development of clinical trials. We have previously demonstrated that intrathecal delivery of scAAV9 in the pig results in efficient transduction of MNs and thus is an alternative to intravenous delivery. We now propose (1) to create a large animal model of SMA by knocking down SMN in MNs with a scAAV9 vector expressing shRNA (2) to correct the SMA phenotype using a second injection of scAAV9 vector expressing hSMN. We injected 5-day old piglets with a single intrathecal dose of scAAV9-shRNA (6.5×10e12vg/kg, n = 6). A few weeks following injection, piglets developed symptoms resembling SMA (abnormal gait and posture) with hind limbs being particularly affected. The phenotype progressed rapidly to a general weakness with posterior paralysis. EMG analysis showed evidence of an active denervation process. We also observed a decrease in both the compound muscle action potential amplitude and the estimated number of MNs. This is remarkably similar to what is observed in SMA patients. Next, we investigated correction of the SMA phenotype. 5-day old piglets (n = 4) received both scAAV9-shRNA (6.5×10e12 vg/kg) and scAAVV9-hSMN (8×10e12 vg/kg) with a 24hrs interval between injections. Two piglets showed no phenotype whereas the other two developed weakness that did not progress. Importantly, no EMG abnormality was observed. We have demonstrated that knockdown of SMN in the pig results in a phenotype displaying the key features of SMA. Furthermore we showed that early reintroduction of SMN significantly alters the phenotype. We are now determining whether later reintroduction of SMN at the first sign of symptoms has a therapeutic benefit as this more closely mimics what will occur in a human clinical trial.

Spinal muscular atrophy (SMA) is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes) that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO) that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.

Spinal muscular atrophy is caused by loss of SMN1 and retention of SMN2. This results in low SMN levels. The SMN protein functions in assembling of Sm proteins onto snRNAs this stabilizes the snRNA and results in transport of the snRNP into the nucleus where they are critical in splicing of genes. While there is currently a debate on whether snRNP assembly or some unknown function in axons is responsible for SMA. We have found direct correlation of snRNP assembly and SMA severity in SMA mice. Furthermore we find no axonal or axon patterning defects in mice and SMN missense mutations that do correct axonal defects in SMN reduced Zebrafish do correct SMA mice and snRNP assembly in these mice. Thus currently we favor disruption of splicing of a select number of target genes in motor neurons as the molecular mechanism. In this regards we have performed laser microdisection of motor neurons from SMA mice and identified a limited number of changes (20) which are specific to SMN deficiency and not due to the state of the motor neuron. These changes are specific to motor neurons and not detectable in total spinal cord extracts we are currently investigating whether restoration gives rescue of the SMA phenotype thus indicating these changes as alternative therapeutic targets. We have also found that mild C and N terminal mutation complement each other and completely rescue Smn-/- mice with no SMA phenotype indicating that there are critical proteins bound by the N and C terminus for the function of SMN in SMA. The SMN function disrupted in SMA is unlikely to be the axonal complex. We are looking at specific back mutation to determine conclusively whether Sm assembly is the critical function affected in SMA. Lastly we have optimized antisense oligonucleotide treatment of SMA by treating at P0-P3 followed by re-administration of the ASO at 30days in a formulation that allows wide spread distribution of the morpholino this results in extended survival beyond 150 days.