Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes

Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% cases of SMA result from deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, does not compensate for the loss of SMN1due to predominant skipping of exon 7. However, correction of SMN2 exon 7 splicing has proven to confer therapeutic benefits in SMA patients. The only approved drug for SMA is an antisense oligonucleotide (Spinraza™/Nusinersen), which corrects SMN2 exon 7 splicing by blocking intronic splicing silencer N1 (ISS-N1) located immediately downstream of exon 7. ISS-N1 is a complex regulatory element encompassing overlapping negative motifs and sequestering a cryptic splice site. More than 40 protein factors have been implicated in the regulation of SMN exon 7 splicing. There is evidence to support that multiple exons of SMN are alternatively spliced during oxidative stress, which is associated with a growing number of pathological conditions. Here, we provide the most up to date account of the mechanism of splicing regulation of the SMN genes

The football is medicine plaform-scientific evidence, large-scale implementation of evidence-based concepts and future perspectives

The idea that football can be used as therapy and as a high-intensity and literally breath-taking training regime goes back centuries. To take one prominent example, the French philosopher Voltaire describes in the Book of Fate (1747), how a patient is cured by playing with a sacred football: “… full-blown and carefully covered with the softest Leather. You must kick this Bladder, Sir, once a Day about your Hall for a whole Hour together, with all the Vigour and Activity you possibly can”, “Ogul, upon making the first Experiment, was ready to expire for want of Breath”, “In short, our Doctor in about 8 days Time, performed an absolute Cure. His Patient was as brisk, active and gay, as One in the Bloom of his Youth.”1 Today, Voltaire and his main character, philosopher Zadig, have been proved right: Football is indeed a breath-taking activity and it can be used as therapy. Albeit today's recommendations suggest a lower training frequency, longer training periods and encourage group-based training, and say that any football can be applied

Pre-mRNA Splicing Modulation by Antisense Oligonucleotides

Pre-mRNA splicing, a dynamic process of intron removal and exon joining, is governed by a combinatorial control exerted by overlapping cis-elements that are unique to each exon and its flanking intronic sequences. Splicing cis-elements are usually 4-to-8-nucleotide-long linear motifs that provide binding sites for specific proteins. Pre-mRNA splicing is also influenced by secondary and higher order RNA structures that affect accessibility of splicing cis-elements. Antisense oligonucleotides (ASOs) that block splicing cis-elements and/or affect RNA structure have been shown to modulate splicing in vivo. Therefore, ASO-based strategies have emerged as a powerful tool for therapeutic manipulation of splicing in pathological conditions. Here we describe an ASO-based approach to increase the production of the full-length SMN2 mRNA in spinal muscular atrophy patient cells

Morpholino-Mediated Exon Inclusion for SMA

The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purpose involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both preclinical studies and clinical trials in spinal muscular atrophy (SMA), which has led to a recently approved drug. AONs with phosphorodiamidate morpholino oligomer (PMO) backbone have shown target engagement with restoration of the defective protein in Duchenne muscular dystrophy (DMD) and their safety profile lead to a recent conditional approval for one DMD PMO drug. PMO AONs are also effective in correcting SMN2 exon 7 splicing and rescuing SMA transgenic mice. Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription (RT-) PCR and quantitative real-time PCR. In addition, we present methodology for protein quantification using western blot in mouse tissues, on neuropathology assessment of skeletal muscle (muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (righting reflex)