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.
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