Evaluation of exon-skipping strategies for Duchenne muscular dystrophy utilizing dystrophin-deficient zebrafish.
Bottom Line: Although this approach has been shown to be effective to restore partially functional dystrophin protein, the level of dystrophin protein that is necessary to rescue a severe muscle pathology has not been addressed.As zebrafish dystrophin mutants (dmd) resemble the severe muscle pathology of human patients, we have utilized this model to evaluate exon skipping.Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration.
Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.Show MeSH
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Mentions: An phosphordiamidate morpholino oligomer, Z32A(–18+7), was designed to target the 5′-end of exon 32. To avoid nonspecific morpholino effects at high concentrations, various concentrations of Z32A(–18 + 7) were administered. By comparison of abnormal embryo rates versus skipping efficiency, the optimal concentration of Z32A(–18 + 7) for injections was established to be 500 μM (data not shown), a concentration which induced a slight curve in the angle of the body axis and a small delay in development. Two days after delivery of Z32A(–18 + 7) into WT embryos, RNA was isolated and RT-PCR across exons 30–34 performed. Although RT-PCR on uninjected embryos led to detection of only WT dystrophin transcript, RT-PCR after injection of Z32A(–18 + 7) resulted in two additional transcripts: one corresponding to a larger transcript arising from insertion of intron 31 upstream and one corresponding to a shorter amplicon originating from simultaneous skipping of the two exons 32 and 33 (Fig. 2A and B). The latter amplicon emanates from a dystrophin transcript with a preserved open reading frame encoding for a shorter dystrophin protein missing 121 amino acids encoded by exons 32 and 33. Interestingly, the proportion of the two Z32A(–18 + 7)-induced transcripts in relation to all detected dystrophin transcripts is significantly higher in dmdpc2/pc2 homozygotes (82%± 0.6%) than in WT embryos (45%± 0.5%; P < 0.01, n = 3), suggesting that the stop mutation-containing transcript could well undergo nonsense mediated decay (Fig. 2F). In addition, even though the amount of the two additional transcripts was declining, they could still be detected 7 days after Z32A(–18 + 7) delivery (Fig. 2F).
Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.