Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon.

Abstract

Several strategies have been pursued to increase the extent of exon 7 inclusion during splicing of SMN2 (survival of motor neuron 2) transcripts, for eventual therapeutic use in spinal muscular atrophy (SMA), a genetic neuromuscular disease. Antisense oligonucleotides (ASOs) that target an exon or its flanking splice sites usually promote exon skipping. Here we systematically tested a large number of ASOs with a 2'-O-methoxy-ethyl ribose (MOE) backbone that hybridize to different positions of SMN2 exon 7, and identified several that promote greater exon inclusion, others that promote exon skipping, and still others with complex effects on the accumulation of the two alternatively spliced products. This approach provides positional information about presumptive exonic elements or secondary structures with positive or negative effects on exon inclusion. The ASOs are effective not only in cell-free splicing assays, but also when transfected into cultured cells, where they affect splicing of endogenous SMN transcripts. The ASOs that promote exon 7 inclusion increase full-length SMN protein levels, demonstrating that they do not interfere with mRNA export or translation, despite hybridizing to an exon. Some of the ASOs we identified are sufficiently active to proceed with experiments in SMA mouse models.