Abstract for presentation at 11th International Congress of Human Genetics

Antisense-mediated correction of spinal muscular atrophy gene through a novel intronic splicing silencer

  • Dr Nirmal Singh, Department of Medicine, UMASS Medical School, Worcester, MA 01605, USA, United States
  • Dr Natalia Singh, Department of Medicine, UMASS Medical School, Worcester, MA 01605, USA, United States
  • Yan Wang, Department of Medicine, UMASS Medical School, Worcester, MA 01605, USA, United States
  • Dr Elliot Androphy, Department of Medicine, UMASS Medical School, Worcester, MA 01605, USA, United States
  • Dr Ravindra Singh, Department of Medicine, UMASS Medical School, Worcester, MA 01605, USA, United States

    • Humans have two near identical copies of the Survival of Motor Neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to an inhibitory mutation at position 6 (C6U mutation in transcript) of exon 7. C6U causes exon 7 skipping during pre-mRNA splicing of SMN2. This leads to production of a truncated protein, which is unstable. For several years, various laboratories are engaged in identifying therapeutic targets to increase the inclusion of exon 7 in SMN2 mRNA. However, very limited success has been achieved because gene-specific targets are inherently difficult to identify. Here we describe a novel silencer element that could be used as a very specific target to correct SMN2 splicing in SMA. We call this element "Intronic Splicing Silencer-N1" (ISS-N1), which is located within the last intron (intron 7) of SMN genes. Deletion of ISS-N1 promoted exon 7 inclusion in mRNAs derived from SMN2 minigene. Underlining the dominant role of ISS-N1 on exon 7 skipping, abrogation of a number of positive cis-elements was tolerated when ISS-N1 was deleted. Confirming the silencer function of ISS-N1, an antisense oligonucleotide against ISS-N1 restored exon 7 inclusion in mRNAs derived from SMN2 minigene or from endogenous SMN2. Consistently, this oligonucleotide increased the levels of SMN protein in SMA-patient-derived cells that carry only SMN2. Remarkably, the increased exon 7 inclusion in SMN2 mRNA was detected at an oligonucleotide concentration as low as 5 nM. This demonstrates ISS-N1 as one of the highly accessible antisense target reported in the literature. Our findings underscore for the first time the profound impact of an evolutionary non-conserved intronic element on SMN2 exon 7 splicing. Significance of ISS-N1 as an efficient therapeutic target for the antisense-oligonucleotide-mediated correction of SMN2 splicing in SMA will be presented.

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