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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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Evaluation of the efficacy of exon skipping for phenotypic rescue of dystrophin-deficient zebrafish. (A) Schematic diagram of the pre-mature dystrophin transcript with exons represented by boxes and introns (not to scale) marked by arches. The positions of the primers (34cDNA, 30For and 34Rev) used for RT-PCR are indicated and green bars mark the position of the morpholino targets. The sizes of exons as well as RT-PCR amplicons are shown in bp and the position of the dmdpc2 mutation is indicated by an asterisk. Blue exons represent translated coding sequence, whereas white exons are not translated with the first stop codon represented by a red block. (B–E) Schematic diagrams of transcripts induced by the indicated morpholinos with the size of the RT-PCR amplicons indicated. (F) Injection of Z32A(–18 + 7) induces a 643 bp and a 219 bp amplicon representing dystrophin transcripts schematically depicted in (B). Interestingly, the proportion of induced exon-skipped transcripts is, with high significance, increased in 2-dpf-old dmdpc2/pc2 homozygotes compared to WT control embryos (82%± 0.6% versus 45%± 0.5%, P < 0.01, n = 3) suggesting that nonsense-mediated decay proportionally reduced the levels of un-skipped stop codon containing transcript. (G) Administration of Z32E(+133+157) leads to exclusion of 78 bp from the 3′-end of exon 32 and administration of Z32E(+83+107) induces inclusion of the intron located upstream of exon 32. Combination of these two morpholinos, however, leads to a robust skipping of exon 32, which is still detected 21 days after administration. Again, the proportion of morpholino-induced transcript is significantly higher in 2-dpf-old dmdpc2/pc2 homozygotes than WT embryos (93%± 2% versus 66%± 3%, P < 0.01, n = 3). (H) Comparison of the zebrafish cryptic splice site elicited by Z32E(+133+157) with the corresponding human dystrophin sequence and the human 5′ splice consensus [24]. ** indicates P < 0.01.
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fig02: Evaluation of the efficacy of exon skipping for phenotypic rescue of dystrophin-deficient zebrafish. (A) Schematic diagram of the pre-mature dystrophin transcript with exons represented by boxes and introns (not to scale) marked by arches. The positions of the primers (34cDNA, 30For and 34Rev) used for RT-PCR are indicated and green bars mark the position of the morpholino targets. The sizes of exons as well as RT-PCR amplicons are shown in bp and the position of the dmdpc2 mutation is indicated by an asterisk. Blue exons represent translated coding sequence, whereas white exons are not translated with the first stop codon represented by a red block. (B–E) Schematic diagrams of transcripts induced by the indicated morpholinos with the size of the RT-PCR amplicons indicated. (F) Injection of Z32A(–18 + 7) induces a 643 bp and a 219 bp amplicon representing dystrophin transcripts schematically depicted in (B). Interestingly, the proportion of induced exon-skipped transcripts is, with high significance, increased in 2-dpf-old dmdpc2/pc2 homozygotes compared to WT control embryos (82%± 0.6% versus 45%± 0.5%, P < 0.01, n = 3) suggesting that nonsense-mediated decay proportionally reduced the levels of un-skipped stop codon containing transcript. (G) Administration of Z32E(+133+157) leads to exclusion of 78 bp from the 3′-end of exon 32 and administration of Z32E(+83+107) induces inclusion of the intron located upstream of exon 32. Combination of these two morpholinos, however, leads to a robust skipping of exon 32, which is still detected 21 days after administration. Again, the proportion of morpholino-induced transcript is significantly higher in 2-dpf-old dmdpc2/pc2 homozygotes than WT embryos (93%± 2% versus 66%± 3%, P < 0.01, n = 3). (H) Comparison of the zebrafish cryptic splice site elicited by Z32E(+133+157) with the corresponding human dystrophin sequence and the human 5′ splice consensus [24]. ** indicates P < 0.01.

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).


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)

Evaluation of the efficacy of exon skipping for phenotypic rescue of dystrophin-deficient zebrafish. (A) Schematic diagram of the pre-mature dystrophin transcript with exons represented by boxes and introns (not to scale) marked by arches. The positions of the primers (34cDNA, 30For and 34Rev) used for RT-PCR are indicated and green bars mark the position of the morpholino targets. The sizes of exons as well as RT-PCR amplicons are shown in bp and the position of the dmdpc2 mutation is indicated by an asterisk. Blue exons represent translated coding sequence, whereas white exons are not translated with the first stop codon represented by a red block. (B–E) Schematic diagrams of transcripts induced by the indicated morpholinos with the size of the RT-PCR amplicons indicated. (F) Injection of Z32A(–18 + 7) induces a 643 bp and a 219 bp amplicon representing dystrophin transcripts schematically depicted in (B). Interestingly, the proportion of induced exon-skipped transcripts is, with high significance, increased in 2-dpf-old dmdpc2/pc2 homozygotes compared to WT control embryos (82%± 0.6% versus 45%± 0.5%, P < 0.01, n = 3) suggesting that nonsense-mediated decay proportionally reduced the levels of un-skipped stop codon containing transcript. (G) Administration of Z32E(+133+157) leads to exclusion of 78 bp from the 3′-end of exon 32 and administration of Z32E(+83+107) induces inclusion of the intron located upstream of exon 32. Combination of these two morpholinos, however, leads to a robust skipping of exon 32, which is still detected 21 days after administration. Again, the proportion of morpholino-induced transcript is significantly higher in 2-dpf-old dmdpc2/pc2 homozygotes than WT embryos (93%± 2% versus 66%± 3%, P < 0.01, n = 3). (H) Comparison of the zebrafish cryptic splice site elicited by Z32E(+133+157) with the corresponding human dystrophin sequence and the human 5′ splice consensus [24]. ** indicates P < 0.01.
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Related In: Results  -  Collection

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fig02: Evaluation of the efficacy of exon skipping for phenotypic rescue of dystrophin-deficient zebrafish. (A) Schematic diagram of the pre-mature dystrophin transcript with exons represented by boxes and introns (not to scale) marked by arches. The positions of the primers (34cDNA, 30For and 34Rev) used for RT-PCR are indicated and green bars mark the position of the morpholino targets. The sizes of exons as well as RT-PCR amplicons are shown in bp and the position of the dmdpc2 mutation is indicated by an asterisk. Blue exons represent translated coding sequence, whereas white exons are not translated with the first stop codon represented by a red block. (B–E) Schematic diagrams of transcripts induced by the indicated morpholinos with the size of the RT-PCR amplicons indicated. (F) Injection of Z32A(–18 + 7) induces a 643 bp and a 219 bp amplicon representing dystrophin transcripts schematically depicted in (B). Interestingly, the proportion of induced exon-skipped transcripts is, with high significance, increased in 2-dpf-old dmdpc2/pc2 homozygotes compared to WT control embryos (82%± 0.6% versus 45%± 0.5%, P < 0.01, n = 3) suggesting that nonsense-mediated decay proportionally reduced the levels of un-skipped stop codon containing transcript. (G) Administration of Z32E(+133+157) leads to exclusion of 78 bp from the 3′-end of exon 32 and administration of Z32E(+83+107) induces inclusion of the intron located upstream of exon 32. Combination of these two morpholinos, however, leads to a robust skipping of exon 32, which is still detected 21 days after administration. Again, the proportion of morpholino-induced transcript is significantly higher in 2-dpf-old dmdpc2/pc2 homozygotes than WT embryos (93%± 2% versus 66%± 3%, P < 0.01, n = 3). (H) Comparison of the zebrafish cryptic splice site elicited by Z32E(+133+157) with the corresponding human dystrophin sequence and the human 5′ splice consensus [24]. ** indicates P < 0.01.
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).

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