Abstracts7th Annual Meeting of the Oligonucleotide Therapeutics SocietyCopenhagen, DenmarkSeptember 8–10, 2011

Abstract

The use of Antisense Oligonucleotides (ASOs) for re-directing splicing has shown promising results as a therapeutic approach for several diseases. A showcase for this technology comes from work done on the Survival of Motor Neuron 2 (SMN2) gene. SMN2 only generates a small amount of full-length mRNA due to a point mutation in exon 7 that causes it to be predominantly skipped. We have previously identified an 18mer 2′-O-methoxyethyl (MOE) modified ASO that promotes almost complete exon 7 inclusion by targeting ISS-N1, an hnRNP A1/A2-dependent intronic splicing silencer in intron 7. We have extended these findings by evaluating how various chemical modifications of the nucleotide sugar moiety of the 18mer ASO affect SMN2 exon 7 splicing. Unexpectedly, we found that incorporating fluorine at the 2′ position (2′-F) of the ribose sugar caused almost complete exon 7 skipping. This skipping activity is dependent on hybridization to the target pre-mRNA and is still robust when the ASO binding is shifted 3′. Furthermore, the 2′-F ASO causes skipping of exon 7 on chromatin associated RNA. We have explored the mechanisms by which the MOE ASO causes SMN2 exon 7 inclusion and the 2′-F ASO causes exon 7 skipping, by doing pull-down experiments with SMN2 RNA immobilized on magnetic beads. We found that the MOE ASO displaces hnRNPA1/A2 from ISS-N1. The 2′-F ASO is still able to displace hnRNPA1/A2, but the resulting 2′-F ASO-RNA duplex is recognized by Interleukin Enhancer Binding Factor 2 and 3 (ILF2 and ILF3). To determine if ILF2 and ILF3 are responsible for the skipping of exon 7, we reduced their expression with siRNAs. Indeed, reduction of ILF2 and ILF3 expression reversed the 2′-F ASO-mediated exon skipping. When administered to SMN2 transgenic mice, the 2′-F ASO caused SMN2 exon 7 skipping in a variety of tissues. These results demonstrate that ASOs with different chemical modifications can be used to promote exon inclusion or exon skipping by either displacing proteins from the pre-mRNA or by recruiting specific proteins to the heteroduplex.