Antisense oligonucleotides and Exon Skipping: Does size matter?
Harding, P.L., Fletcher, S. and Wilton, S.D. (2005) Antisense oligonucleotides and Exon Skipping: Does size matter? In: 4th Australasian Gene Therapy Society Meeting, 27 - 29 April 2005, Rydges Hotel, Carlton, VIC.
Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder that arises from mutations in the dystrophin gene. The disease is characterised by severe and progressive wasting of skeletal and cardiac muscles. The mdx mouse model of muscular dystrophy has a nonsense mutation in exon 23 of the dystrophin gene. Our group has previously shown that both 2_-O-methyl antisense oligonucleotides (AOs) and morpholino AOs can induce skipping of exon 23 in H2k-mdx cells. These AOs redirect splicing by masking the normal donor splice site to induce exon 23 skipping, causing an in-frame transcript to be produced which can then be translated into a shorter but functional dystrophin protein. We have compared 2_-O-methyl AOs with morpholino AOs, to determine which chemistry is the most efficient at inducing exon skipping and presumably which chemistry should be used in human clinical trials. Two different sequences targeting the intron 23 donor splice site, each prepared as 2_- O-methyl or morpholino AOs, were evaluated to determine if the length of the AO influences efficiency of exon skipping. Both morpholino and 2_-O-methyl AOs were used to transfect H2k-mdx cells across a range of concentrations. Persistence of corrected in-frame transcript was also used as ameasure to determine the most efficient AO for inducing exon skipping. We found the longer AO for each chemistry was more efficient than the corresponding shorter compound. The morpholino AOs, when delivered as a leash-lipoplex, were more efficient at inducing sustained exon skipping than the corresponding sequences prepared as 2_-O-methyl AOs in vitro. Both chemistries are now being evaluated in vivo for safety and efficacy.
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