title: Dual masking of specific negative splicing regulatory elements resulted in maximal exon 7 inclusion of SMN2 gene.
abstract: Spinal muscular atrophy (SMA) is a fatal autosomal recessive disease caused by survival motor neuron (SMN) protein insufficiency due to SMN1 mutations. Boosting SMN2 expression is a potential therapy for SMA. SMN2 has identical coding sequence as SMN1 except for a silent C-to-T transition at the 6th nucleotide of exon 7, converting a splicing enhancer to a silencer motif. Consequently, most SMN2 transcripts lack exon 7. More than ten putative splicing regulatory elements (SREs) were reported to regulate exon 7 splicing. To investigate the relative strength of each negative SRE in inhibiting exon 7 inclusion, antisense oligonucleotides (AONs) were used to mask each element, and the fold increase of full-length SMN transcripts containing exon 7 were compared. The most potent negative SREs are at intron 7 (in descending order): ISS-N1, 3' splice site of exon 8 (ex8 3'ss) and ISS+100. Dual-targeting AONs were subsequently used to mask two nonadjacent SREs simultaneously. Notably, masking of both ISS-N1 and ex8 3'ss induced the highest fold increase of full-length SMN transcripts and proteins. Therefore, efforts should be directed towards the two elements simultaneously for the development of optimal AONs for SMA therapy.
authors: Formerly Department of Clinical Research, Singapore General Hospital, Singapore, Singapore.
authors: Peng Wen
authors: PW
authors: Pao
authors: A*STAR Institute of High Performance Computing, Singapore, Singapore.
authors: Keng Boon
authors: KB
authors: Wee
authors: Formerly Department of Clinical Research, Singapore General Hospital, Singapore, Singapore.
authors: Woon Chee
authors: WC
authors: Yee
authors: Department of Research, National Skin Centre, Singapore, Singapore.
authors: Zacharias Aloysius Dwi
authors: ZA
authors: Pramono
authors: Department of Research, National Skin Centre, Singapore, Singapore.
authors: Zacharias Aloysius
authors: ZA
authors: Dwipramono
mesh_terms: D004742:Enhancer Elements, Genetic / Q000235:genetics; D005091:Exons / Q000235:genetics; D015316:Genetic Therapy; D006801:Humans; D009046:Motor Neurons / Q000378:metabolism / Q000473:pathology; D009134:Muscular Atrophy, Spinal / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D022821:RNA Splice Sites; D012326:RNA Splicing / Q000235:genetics*; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics; D055540:Survival of Motor Neuron 2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000627:therapeutic use; D014158:Transcription, Genetic
chemical_list: D016376:Oligonucleotides, Antisense; D022821:RNA Splice Sites; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein