title: Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice.
abstract: 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.
authors: Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA.
authors: Yimin
authors: Y
authors: Hua
authors: Timothy A
authors: TA
authors: Vickers
authors: Hazeem L
authors: HL
authors: Okunola
authors: C Frank
authors: CF
authors: Bennett
authors: Adrian R
authors: AR
authors: Krainer
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D005091:Exons; D015316:Genetic Therapy; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism*; D006801:Humans; D007438:Introns; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009134:Muscular Atrophy, Spinal / Q000628:therapy; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use; D012326:RNA Splicing / Q000187:drug effects*; D016601:RNA-Binding Proteins / Q000235:genetics*; D055532:SMN Complex Proteins; D055540:Survival of Motor Neuron 2 Protein
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526477:SMN2 protein, human; D055540:Survival of Motor Neuron 2 Protein; C439768:hnRNP A2