An Investigation into the Mechanism of Action of the SMN2 Bifunctional Oligonucleotide and its Application to Reverse the RON Δ165 Pro-metastatic Splicing Event
thesisposted on 08.06.2012, 14:16 by Lindsay Denise Smith
It is widely accepted that alternative splicing is often a catalyst for the development and progression of disease. Recent advances in oligonucleotide therapeutics have seen these alternative splicing patterns forcibly changed, providing a specific and versatile attack against such diseases. This study has focused on the use of bifunctional oligonucleotides for the treatment of spinal muscular atrophy (SMA) and cancer. In SMA the bifunctional oligonucleotide is targeted to inhibit exon 7 skipping during splicing of SMN2. The current study has confirmed that this oligonucleotide is able to stimulate SMN2 exon 7 splicing, as has been seen previously (Skordis et al., 2003; Owen et al., 2011). Analysis of the action of this oligonucleotide indicated that it specifically recruits spliceosomal factors to the 3’ splice site of SMN2 intron 6. The tail domain was seen to enhance binding of U2 snRNP to this region and stabilize protein complex formation, while the annealing domain was seen to enhance U2AF65-RNA binding. A greater understanding of the mechanism of action of the SMN2 bifunctional oligonucleotide also highlighted flaws in the design of the previously tested RON exon 11 bifunctional oligonucleotide (Ghigna et al., 2010). This oligonucleotide was designed to combat the production of RON Δ165 mRNA, in which RON exon 11 is skipped. Expression of this RON isoform has been linked with the onset of metastasis in some epithelial cell cancers. A systematic analysis of the splicing characteristics of this region in RON pre-mRNA, suggested that weak 3’ splice site sequences limit RON exon 11 splicing. hnRNP H was also shown to bind RON introns 10 and 11 and may play a role in the regulation of RON exon 11 splicing. A new bifunctional oligonucleotide targeted combat RON Δ165 mRNA production, through the stimulation of RON exon 11 splicing, was designed with the weakness in RON exon 11 splicing and the mechanism of action of the SMN2 bifunctional oligonucleotide in mind.