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: For this purpose, we in-crossed dmdpc2/+ carriers and injected the offspring with Z32E(+133+157) and Z32E(+83+107) combined at an equal concentration of 12 μM. First, to test the accuracy of the morpholino administration, dmdpc2/pc2 larvae were identified by PCR and subsequently analysed for their induced skipping effect individually. As shown in Figure S3, the variation in the detected skipping effects was relatively low. Therefore, in subsequent experiments with morpholino concentrations varying from 0 to 12 μM, two dmdpc2/pc2 homozygous larvae were combined and the proportion of exon 32 skipped transcript was analysed in relation to all dystrophin transcripts by RT-PCR. Also, progression of the dystrophic pathology after each morpholino administration was assessed by analysis of the muscle birefringence at 3 dpf (Fig. 4). The extremes of the injected concentrations, 0 and 12 μM, resulted in a Mendelian ratio of 24.6%± 0.5% of affected larvae and no detectable dystrophic phenotype, respectively (data in mean ± S.E.M.). Intermediate concentrations, however, resulted in partial rescue of the birefringence in a highly dose-dependent manner as manifested by intermediate phenotype ratios. Collected data suggest that a skipping efficiency of about 10% results in 10% of detected dystrophic fish, representing a reduction of the expected Mendalian ratio by about half. In contrast to this partial rescue, a skipping efficiency of about 30–40% seems to result in less than 3% of injected larvae with a detectable dystrophic pathology, revealing that a skipping efficiency of 30–40% is sufficient to evoke a near full rescue of the dystrophic phenotype. As described above, injection of Z32E(+133+157) and Z32E(+83+107) into WT embryos resulted in lower exon-skipping efficiencies. Therefore, skipping efficiencies measured in homozygous mutants were also analysed in WT embryos. Although exon skipping could not be detected at morpholino concentrations of 0 μM, 1 μM and 2 μM, injection of 12 μM of the morpholino combination resulted in 12%± 1% skipping efficiency, 6 μM in 3.5%± 0.2%, 5 μM in 2.8%± 0.3%, 4 μM in 1.5%± 0.2% and 3 μM in 0.6%± 0.1% efficiency.
Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.