T.P.2.01 Antisense oligomer design: Targeting and assay systems
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Antisense oligonucleotides (AOs) can disrupt exon recognition and splicing during intron removal from the mature mRNA. We, and others, have found that approximately two out of three AOs can induce some exon skipping. However, the efficiency of exon removal varies greatly within and between exons. Some AOs may induce weak exon removal only after transfection at high concentrations, while other more optimised compounds induce substantial exon skipping at much lower concentrations. An empirical approach to AO design is to evaluate a ”first pass” panel of AOs to identify the most promising target in cultured human cells, and then design a series of overlapping AOs for further evaluation. Oligomer length and target annealing site are critical parameters in AO design. Despite strong homology between the mouse and human dystrophin genes, and the observation that a normal dystrophin gene is processed correctly in transgenic mice, we have identified several examples where targeting the same AO annealing sites leads to different exon skipping outcomes. Targeting the exon 23 donor splice site leads to efficient exon skipping in the mouse, while the human equivalent site was totally unresponsive. An oligomer designed to excise exon 16 from the normal dystrophin pre-mRNA was found to be more than an order of magnitude more efficient when applied to a dystrophic cell line with an exon 16 splice motif mutation. When such variation in AO efficacy is observed in cell lines expressing different dystrophin transcripts, the validity of using artificial systems, such as a plasmid containing a single dystrophin exon in the context of limited flanking sequence in a reporter gene, for oligomer design must be questioned. We propose that the most valid system in which to evaluate clinically relevant AOs is one in which dystrophin mRNA is processed in the presence of all normal cis and trans splicing elements.
|Publication Type:||Journal Article|
|Copyright:||© 2008 Published by Elsevier B.V.|
|Notes:||Abstract: 13th International Congress of the World Muscle Society, Newcastle Upon Tyne, UK. 29 September - 2 October 2008|
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