Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3 ' splice site pairing

Spinal muscular atrophy is caused by the loss of functional survival motor neuron (SMN1) alleles. A translationally silent nucleotide transition in th e duplicated copy of the gene (SMN2) leads to exon 7 skipping and expressio n of a nonfunctional gene product. It has been suggested that differential SMN2 splicing is caused by the disruption of an exonic splicing enhancer. H ere we show that the single nucleotide difference reduces the intrinsic str ength of the 3' splice site of exon 7 2-fold, whereas the strength of the 5 ' splice site of the exon 7 is not affected. Thus, a decrease in splice sit e strength is magnified in the context of competing exons. These data sugge st that lower levels of exon 7 definition not only reduce intron 6 removal but, more importantly, increase the efficiency of the competing exon 7 skip ping pathway. Antisense oligonucleotides were tested to modulate exon 7 inc lusion, which contains the authentic translation stop codon. Oligonucleotid es directed toward the 3' splice site of exon 8 were shown to alter SMN2 sp licing in favor of exon 7 inclusion. These results suggest that antisense o ligonucleotides could be used as a therapeutic strategy to counteract the p rogression of SMA.