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.
| Singh, Natalia N; Luo, Diou; Singh, Ravindra N (2018) Pre-mRNA Splicing Modulation by Antisense Oligonucleotides. Methods Mol Biol 1828:415-437 |
| Singh, Ravindra N; Howell, Matthew D; Ottesen, Eric W et al. (2017) Diverse role of survival motor neuron protein. Biochim Biophys Acta Gene Regul Mech 1860:299-315 |
| Singh, Natalia N; Lee, Brian M; DiDonato, Christine J et al. (2015) Mechanistic principles of antisense targets for the treatment of spinal muscular atrophy. Future Med Chem 7:1793-808 |
| Howell, Matthew D; Singh, Natalia N; Singh, Ravindra N (2014) Advances in therapeutic development for spinal muscular atrophy. Future Med Chem 6:1081-99 |
| Sivanesan, Senthilkumar; Howell, Matthew D; Didonato, Christine J et al. (2013) Antisense oligonucleotide mediated therapy of spinal muscular atrophy. Transl Neurosci 4: |
| Singh, Natalia N; Shishimorova, Maria; Cao, Lu Cheng et al. (2009) A short antisense oligonucleotide masking a unique intronic motif prevents skipping of a critical exon in spinal muscular atrophy. RNA Biol 6:341-50 |
| Papp, Laura V; Wang, Junning; Kennedy, Derek et al. (2008) Functional characterization of alternatively spliced human SECISBP2 transcript variants. Nucleic Acids Res 36:7192-206 |
| Singh, Ravindra N (2007) Evolving concepts on human SMN pre-mRNA splicing. RNA Biol 4:7-10 |
| Singh, Ravindra N (2007) Unfolding the mystery of alternative splicing through a unique method of in vivo selection. Front Biosci 12:3263-72 |
| Singh, Ravindra N (2006) Association of severity of spinal muscular atrophy with the loss of NAIP gene. Neurol India 54:246 |
