Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice

Am J Hum Genet. 2008 Apr;82(4):834-48. doi: 10.1016/j.ajhg.2008.01.014. Epub 2008 Mar 27.


Survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Cyclic AMP Response Element-Binding Protein / genetics*
  • Exons
  • Genetic Therapy
  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B / metabolism*
  • Humans
  • Introns
  • Mice
  • Mice, Transgenic
  • Molecular Sequence Data
  • Muscular Atrophy, Spinal / therapy
  • Nerve Tissue Proteins / genetics*
  • Oligonucleotides, Antisense / genetics
  • Oligonucleotides, Antisense / pharmacology*
  • Oligonucleotides, Antisense / therapeutic use
  • RNA Splicing / drug effects*
  • RNA-Binding Proteins / genetics*
  • SMN Complex Proteins
  • Survival of Motor Neuron 2 Protein


  • Cyclic AMP Response Element-Binding Protein
  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B
  • Nerve Tissue Proteins
  • Oligonucleotides, Antisense
  • RNA-Binding Proteins
  • SMN Complex Proteins
  • SMN2 protein, human
  • Survival of Motor Neuron 2 Protein
  • hnRNP A2