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Evaluation of exon-skipping strategies for Duchenne muscular dystrophy utilizing dystrophin-deficient zebrafish.

Berger J, Berger S, Jacoby AS, Wilton SD, Currie PD - J. Cell. Mol. Med. (2011)

Bottom Line: By utilizing antisense oligonucleotides, splicing of the dystrophin transcript can be altered so that exons harbouring a mutation are excluded from the mature mRNA.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.Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration.

View Article: PubMed Central - PubMed

Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

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Related in: MedlinePlus

Determination of phenotypic rescue threshold for exon-skipping efficiency. Different concentrations of an equal mixture of Z32E(+133+157) and Z32E(+83+107) were injected into embryos of an in-cross of heterozygous dmdpc2/+ fish. At 3 days post administration, resulting levels of exon-skipping efficiency (blue line) were evaluated by RT-PCR and depicted as the percentage of skipped transcript out of total dystrophin transcript. The corresponding level of phenotypic rescue, as evaluated by the birefringence assay, is displayed as the percentage of larvae possessing a dystrophic phenotype from all injected larvae. Administration of doses of combined Z32E(+133+157) and Z32E(+83+107) below 2 μM does not change the Mendalian ratio (25%) of detected dystrophic mutants. In contrast, no larvae display a dystrophic phenotype when injected with a concentration of 12 μM and administration of intermediate concentrations lead to partial rescue as specified in the depicted diagram. These data suggest an exon-skipping efficiency of 20–30% is required to induce effective phenotypic rescue of the highly penetrant dystrophic phenotype evident in the zebrafish model. Data are means ± S.E.M.
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fig04: Determination of phenotypic rescue threshold for exon-skipping efficiency. Different concentrations of an equal mixture of Z32E(+133+157) and Z32E(+83+107) were injected into embryos of an in-cross of heterozygous dmdpc2/+ fish. At 3 days post administration, resulting levels of exon-skipping efficiency (blue line) were evaluated by RT-PCR and depicted as the percentage of skipped transcript out of total dystrophin transcript. The corresponding level of phenotypic rescue, as evaluated by the birefringence assay, is displayed as the percentage of larvae possessing a dystrophic phenotype from all injected larvae. Administration of doses of combined Z32E(+133+157) and Z32E(+83+107) below 2 μM does not change the Mendalian ratio (25%) of detected dystrophic mutants. In contrast, no larvae display a dystrophic phenotype when injected with a concentration of 12 μM and administration of intermediate concentrations lead to partial rescue as specified in the depicted diagram. These data suggest an exon-skipping efficiency of 20–30% is required to induce effective phenotypic rescue of the highly penetrant dystrophic phenotype evident in the zebrafish model. Data are means ± S.E.M.

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.


Evaluation of exon-skipping strategies for Duchenne muscular dystrophy utilizing dystrophin-deficient zebrafish.

Berger J, Berger S, Jacoby AS, Wilton SD, Currie PD - J. Cell. Mol. Med. (2011)

Determination of phenotypic rescue threshold for exon-skipping efficiency. Different concentrations of an equal mixture of Z32E(+133+157) and Z32E(+83+107) were injected into embryos of an in-cross of heterozygous dmdpc2/+ fish. At 3 days post administration, resulting levels of exon-skipping efficiency (blue line) were evaluated by RT-PCR and depicted as the percentage of skipped transcript out of total dystrophin transcript. The corresponding level of phenotypic rescue, as evaluated by the birefringence assay, is displayed as the percentage of larvae possessing a dystrophic phenotype from all injected larvae. Administration of doses of combined Z32E(+133+157) and Z32E(+83+107) below 2 μM does not change the Mendalian ratio (25%) of detected dystrophic mutants. In contrast, no larvae display a dystrophic phenotype when injected with a concentration of 12 μM and administration of intermediate concentrations lead to partial rescue as specified in the depicted diagram. These data suggest an exon-skipping efficiency of 20–30% is required to induce effective phenotypic rescue of the highly penetrant dystrophic phenotype evident in the zebrafish model. Data are means ± S.E.M.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4373433&req=5

fig04: Determination of phenotypic rescue threshold for exon-skipping efficiency. Different concentrations of an equal mixture of Z32E(+133+157) and Z32E(+83+107) were injected into embryos of an in-cross of heterozygous dmdpc2/+ fish. At 3 days post administration, resulting levels of exon-skipping efficiency (blue line) were evaluated by RT-PCR and depicted as the percentage of skipped transcript out of total dystrophin transcript. The corresponding level of phenotypic rescue, as evaluated by the birefringence assay, is displayed as the percentage of larvae possessing a dystrophic phenotype from all injected larvae. Administration of doses of combined Z32E(+133+157) and Z32E(+83+107) below 2 μM does not change the Mendalian ratio (25%) of detected dystrophic mutants. In contrast, no larvae display a dystrophic phenotype when injected with a concentration of 12 μM and administration of intermediate concentrations lead to partial rescue as specified in the depicted diagram. These data suggest an exon-skipping efficiency of 20–30% is required to induce effective phenotypic rescue of the highly penetrant dystrophic phenotype evident in the zebrafish model. Data are means ± S.E.M.
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.

Bottom Line: By utilizing antisense oligonucleotides, splicing of the dystrophin transcript can be altered so that exons harbouring a mutation are excluded from the mature mRNA.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.Novel dmd mutations were identified to enable the design of phenotype rescue studies via morpholino administration.

View Article: PubMed Central - PubMed

Affiliation: Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

Show MeSH
Related in: MedlinePlus