Humans have two copies of Survival of Motor Neuron (SMN) gene, SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), a debilitating disease of infants and children. SMN2 fails to compensate for the loss of SMN1 due to skipping of exon 7 resulting in the synthesis of a truncated protein, which is unstable. Presence of SMN2 in most SMA patients provides a rare opportunity to restore SMN levels by correcting the aberrant splicing of exon 7. Using a novel method of in vivo selection, we previously reported that a weak 5'splice site (51 ss) serves as the limiting factor for exon 7 inclusion in SMN2. Based on this observation, we have recently discovered that a unique intronic inhibitory element contributes towards the weak 5'ss of exon 7. We call this element Intronic Splicing Silencer N1 (abbreviated as ISS-N1). Interestingly, ISS-N1 appears to be unique to humans. Based on our preliminary results, ISS-N1 plays an important role in pathogenesis of SMA. Most importantly, we discovered that Antisense Oligonucleotides (abbreviated as ASOs) that targeted ISS-N1 corrected aberrant splicing of SMN2 in all cell types tested including SMA patient cells. Consequently, ASO against ISS-N1 fully restored SMN levels in patient cells. Significantly, the ASO-mediated stimulatory effect was observed even at low ASO doses, suggesting that ISS-N1 is a highly accessible antisense target. The antisense effect was very specific to ISS-N1 as two or more mutations within ISS-N1 completely eliminated the ASO-mediated stimulatory effect. Based on these results we believe that we have discovered an ideal target-site for the ASO-mediated therapy of SMA. In this grant proposal, we will (1) characterize ISS-N1, its RNA structure and interacting factors in details;(2) develop efficient ASOs against ISS-N1 to correct SMN2 splicing in patient cells;and finally (3) conduct in vivo studies, using mice models of SMA to validate ASOs against ISS-N1 as the possible drug candidates. The most frequent cause of spinal muscular atrophy (SMA) is the loss of SMN1 gene accompanied by the inability of SMN2 gene to compensate due to aberrant splicing. Here, we will characterize a novel intronic element that plays a critical role in pathogenesis of SMA. In addition, we will use this element as a target for the antisense-mediated correction of aberrant splicing in SMA.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Cell Death in Neurodegeneration Study Section (CDIN)
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Porter, John D
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Iowa State University
Veterinary Sciences
Schools of Veterinary Medicine
United States
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Ottesen, Eric W (2017) ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy. Transl Neurosci 8:1-6
Ottesen, Eric W; Seo, Joonbae; Singh, Natalia N et al. (2017) A Multilayered Control of the Human Survival Motor Neuron Gene Expression by Alu Elements. Front Microbiol 8:2252
Howell, Matthew D; Ottesen, Eric W; Singh, Natalia N et al. (2017) Gender-Specific Amelioration of SMA Phenotype upon Disruption of a Deep Intronic Structure by an Oligonucleotide. Mol Ther 25:1328-1341
Singh, Natalia N; Del Rio-Malewski, José Bruno; Luo, Diou et al. (2017) Activation of a cryptic 5' splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene. Nucleic Acids Res 45:12214-12240
Howell, Matthew D; Ottesen, Eric W; Singh, Natalia N et al. (2017) TIA1 is a gender-specific disease modifier of a mild mouse model of spinal muscular atrophy. Sci Rep 7:7183
Singh, Ravindra N; Howell, Matthew D; Ottesen, Eric W et al. (2017) Diverse role of survival motor neuron protein. Biochim Biophys Acta 1860:299-315
Singh, N N; Howell, M D; Androphy, E J et al. (2017) How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy. Gene Ther 24:520-526
Seo, Joonbae; Singh, Natalia N; Ottesen, Eric W et al. (2016) A novel human-specific splice isoform alters the critical C-terminus of Survival Motor Neuron protein. Sci Rep 6:30778
Seo, Joonbae; Singh, Natalia N; Ottesen, Eric W et al. (2016) Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene. PLoS One 11:e0154390
Ottesen, Eric W; Howell, Matthew D; Singh, Natalia N et al. (2016) Severe impairment of male reproductive organ development in a low SMN expressing mouse model of spinal muscular atrophy. Sci Rep 6:20193

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