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Multiple exon skipping to correct duplications in the dystrophin gene

Greer, K., Fletcher, S. and Wilton, S.D. (2018) Multiple exon skipping to correct duplications in the dystrophin gene. Journal of Gene Medicine, 20 (1).

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Duchenne Muscular Dystrophy is a severe muscle wasting disease caused by protein truncating mutations in DMD that results in the absence of a functional protein. Exonic deletions are the most common type of DMD lesion, however whole exon duplications account for approximately 10‐15% of all reported mutations. Here we describe in vitro evaluation of two different antisense oligomer chemistries to correct exonic duplications of the DMD gene through splice switching. Phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide and 2'O methyl phosphorothioate antisense oligomers were tested on four Duchenne muscular dystrophy patient cell strains with exon duplications in the actin binding domain. In all patient cell strains, the removal of exons 6‐8 was needed in order to generate an in‐frame transcript. Two PCR systems were compared, one using a DNA polymerase designed for sensitivity, specificity and yield (SSIII/AmpliTaq Gold) and one using a high fidelity PCR enzyme for long range PCR (Takara LA Taq). Apparent differences in exon skipping efficiencies were evident. Takara LA Taq produced more of the full length duplicated transcript in comparison to the SSIII/AmpliTaq Gold amplification system. Due to incurred “PCR slippage”, the SSIII/ Amplitaq Gold system resulted in preferential amplification of the smaller PCR products, as well as substantial amounts of an apparently normal dystrophin transcript. Combinations of phosphorodiamidate morpholino oligomers targeting exons 6‐8 in the patient cell strains were more efficient at restoring a multi‐ exon skipped in‐frame transcript compared to the same combinations of oligomers with the 2'O methyl phosphorothioate chemistry.

Item Type: Journal Article
Murdoch Affiliation(s): Centre for Comparative Genomics
Publisher: John Wiley & Sons
Copyright: © 2018 John Wiley & Sons, Ltd.
Other Information: Abstract taken from the Joint 10th Australasian Gene and Cell Therapy Society (AGCTS) and Australasian Society for Stem Cell Research (ASSCR) Annual Scientific Meeting
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