Targeting a novel silencer to correct SMN2 splicing in Spinal Muscular Atrophy

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The most frequent cause of spinal muscular atrophy (SMA) is the loss of Survival Motor Neuron 1 (SMN1) gene, which produces SMN protein. A nearly identical copy of the gene, SMN2, that produces nonfunctional SMN protein due to skipping of exon 7, fails to compensate for the loss of SMN1. My laboratory investigates regulation of SMN exon 7 splicing with the goal of identification of therapeutic targets to promote exon 7 nclusion during pre-mRNA splicing of SMN2. Using a state of the art method of iterative selection, we have recently shown that skipping of exon 7 in SMN2 is directly linked to the weak 5' splice site (5' ss) of exon 7. Upon extending our investigation, we recently discovered a novel Intronic Splicing Silencer (ISS-N1) that facilitates skipping of exon 7 by sequestering the 5' ss. Supporting the inhibitory nature of ISS-N1, mutations and deletions within ISS-N1 promoted exon 7 inclusion in SMN2 mRNA. Further confirming the inhibitory nature of ISS-N1, the antisense oligo (ASO) that blocked ISS-N1 fully restored exon 7 inclusion in both, our minigene system and SMA patient fibroblasts (from the endogenous SMN2). As a consequence, ASO- treated patient cells showed increased expression of SMN protein from SMN2. 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 ISS-N1 offers a unique target-site for ASO-mediated therapy of SMA. Antisense technology has emerged as a powerful tool to treat many human diseases. This grant proposal is aimed at designing highly efficient ASOs against ISS-N1. We will examine the effect of ASOs in SMA patient cells as well as in mice models of SMA. The findings from this study will establish the efficacy of ASO-based therapy of SMA. 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 use antisense oligos that correct aberrant splicing of SMN2 by targeting a novel intronic silencer that we discovered recently. To explore the therapeutic potential of antisense oligos, we will extend our study to the mice models of SMA.