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Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy.

Kemaladewi DU, Hoogaars WM, van Heiningen SH, Terlouw S, de Gorter DJ, den Dunnen JT, van Ommen GJ, Aartsma-Rus A, ten Dijke P, 't Hoen PA - BMC Med Genomics (2011)

Bottom Line: Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms.In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients.It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, Leiden, 2300RC, the Netherlands.

ABSTRACT

Background: Myostatin is a potent muscle growth inhibitor that belongs to the Transforming Growth Factor-β (TGF-β) family. Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms. By contrast, Duchenne muscular dystrophy (DMD) is characterized by a loss of muscle fibers and impaired regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients.

Methods: We targeted myostatin exon 2 using antisense oligonucleotides (AON) in healthy and DMD-derived myotubes cultures. We assessed the exon skipping level, transcriptional expression of myostatin and its target genes, and combined myostatin and several dystrophin AONs. These AONs were also applied in the mdx mice models via intramuscular injections.

Results: Myostatin AON induced exon 2 skipping in cell cultures and to a lower extent in the mdx mice. It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression. Furthermore, combination of myostatin and dystrophin AONs induced simultaneous skipping of both genes.

Conclusions: We conclude that two AONs can be used to target two different genes, MSTN and DMD, in a straightforward manner. Targeting multiple ligands of TGF-beta family will be more promising as adjuvant therapies for DMD.

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Related in: MedlinePlus

Schematic overview of myostatin and dystrophin exon skipping. The myostatin gene (MSTN) consists of three exons, whereas the protein consists of three domains: signaling (sig.), propeptide and mature domains. The position of each exon relative to each domain is denoted by broken lines (A). Antisense oligonucleotides (AON) targeting exon 2 will hybridize and hide the exon from the splicing machinery, resulting in skipping exon 2 upon mRNA splicing. The removal of exon 2 will disrupt the open reading frame (ORF; *) and the protein will lack part of the propeptide and the entire mature domain (B). Dystrophin gene (DMD) consists of 79 exons, whereas the proteins consist of actin binding-, central rod, cysteine rich- and C-terminal domains (C). One of the examples of DMD is deletion in exon 48-50 which disrupted the reading frame and introduces premature stop codon (*). Due to this mutation, part of the central rod and the entire Cys-rich and C-terminal domains are missing (D). In the therapy (currently in clinical trials), AON is directed towards exon 51. With the similar principle, exon 51 will be skipped upon mRNA splicing which in turn restores the ORF. This internally-deleted DMD will be translated as dystrophin with shorter central rod domain. However, since the essential C-terminal domain is retained, the protein is partially functional (E).
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Figure 1: Schematic overview of myostatin and dystrophin exon skipping. The myostatin gene (MSTN) consists of three exons, whereas the protein consists of three domains: signaling (sig.), propeptide and mature domains. The position of each exon relative to each domain is denoted by broken lines (A). Antisense oligonucleotides (AON) targeting exon 2 will hybridize and hide the exon from the splicing machinery, resulting in skipping exon 2 upon mRNA splicing. The removal of exon 2 will disrupt the open reading frame (ORF; *) and the protein will lack part of the propeptide and the entire mature domain (B). Dystrophin gene (DMD) consists of 79 exons, whereas the proteins consist of actin binding-, central rod, cysteine rich- and C-terminal domains (C). One of the examples of DMD is deletion in exon 48-50 which disrupted the reading frame and introduces premature stop codon (*). Due to this mutation, part of the central rod and the entire Cys-rich and C-terminal domains are missing (D). In the therapy (currently in clinical trials), AON is directed towards exon 51. With the similar principle, exon 51 will be skipped upon mRNA splicing which in turn restores the ORF. This internally-deleted DMD will be translated as dystrophin with shorter central rod domain. However, since the essential C-terminal domain is retained, the protein is partially functional (E).

Mentions: In this study we chose to use 2'OMePS AON to target MSTN exon 2 and disrupt its translational reading frame in order to knockdown its expression level. As it harbors the same chemistry modification as the PRO051 DMD AON currently in trial, we hypothesized that the administration of the two AONs as a cocktail would be achievable and may thus yield a combination treatment by simultaneously correcting the Dmd transcript and downregulating the Mstn transcript. In addition, as recently reviewed in [39], the 2'OMePS modification enhances the stability and increases the in vivo half-life compared to the previously studied butanol-tagged antisense [40] or siRNA-mediated RNA interference approaches targeting myostatin [41]. We evaluated its feasibility to induce exon skipping and downregulate MSTN expression in myotubes cultures and DMD mouse models. Furthermore, we combined it with several DMD AONs to look into the possibility of skipping two genes simultaneously (Figure 1).


Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy.

Kemaladewi DU, Hoogaars WM, van Heiningen SH, Terlouw S, de Gorter DJ, den Dunnen JT, van Ommen GJ, Aartsma-Rus A, ten Dijke P, 't Hoen PA - BMC Med Genomics (2011)

Schematic overview of myostatin and dystrophin exon skipping. The myostatin gene (MSTN) consists of three exons, whereas the protein consists of three domains: signaling (sig.), propeptide and mature domains. The position of each exon relative to each domain is denoted by broken lines (A). Antisense oligonucleotides (AON) targeting exon 2 will hybridize and hide the exon from the splicing machinery, resulting in skipping exon 2 upon mRNA splicing. The removal of exon 2 will disrupt the open reading frame (ORF; *) and the protein will lack part of the propeptide and the entire mature domain (B). Dystrophin gene (DMD) consists of 79 exons, whereas the proteins consist of actin binding-, central rod, cysteine rich- and C-terminal domains (C). One of the examples of DMD is deletion in exon 48-50 which disrupted the reading frame and introduces premature stop codon (*). Due to this mutation, part of the central rod and the entire Cys-rich and C-terminal domains are missing (D). In the therapy (currently in clinical trials), AON is directed towards exon 51. With the similar principle, exon 51 will be skipped upon mRNA splicing which in turn restores the ORF. This internally-deleted DMD will be translated as dystrophin with shorter central rod domain. However, since the essential C-terminal domain is retained, the protein is partially functional (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3107769&req=5

Figure 1: Schematic overview of myostatin and dystrophin exon skipping. The myostatin gene (MSTN) consists of three exons, whereas the protein consists of three domains: signaling (sig.), propeptide and mature domains. The position of each exon relative to each domain is denoted by broken lines (A). Antisense oligonucleotides (AON) targeting exon 2 will hybridize and hide the exon from the splicing machinery, resulting in skipping exon 2 upon mRNA splicing. The removal of exon 2 will disrupt the open reading frame (ORF; *) and the protein will lack part of the propeptide and the entire mature domain (B). Dystrophin gene (DMD) consists of 79 exons, whereas the proteins consist of actin binding-, central rod, cysteine rich- and C-terminal domains (C). One of the examples of DMD is deletion in exon 48-50 which disrupted the reading frame and introduces premature stop codon (*). Due to this mutation, part of the central rod and the entire Cys-rich and C-terminal domains are missing (D). In the therapy (currently in clinical trials), AON is directed towards exon 51. With the similar principle, exon 51 will be skipped upon mRNA splicing which in turn restores the ORF. This internally-deleted DMD will be translated as dystrophin with shorter central rod domain. However, since the essential C-terminal domain is retained, the protein is partially functional (E).
Mentions: In this study we chose to use 2'OMePS AON to target MSTN exon 2 and disrupt its translational reading frame in order to knockdown its expression level. As it harbors the same chemistry modification as the PRO051 DMD AON currently in trial, we hypothesized that the administration of the two AONs as a cocktail would be achievable and may thus yield a combination treatment by simultaneously correcting the Dmd transcript and downregulating the Mstn transcript. In addition, as recently reviewed in [39], the 2'OMePS modification enhances the stability and increases the in vivo half-life compared to the previously studied butanol-tagged antisense [40] or siRNA-mediated RNA interference approaches targeting myostatin [41]. We evaluated its feasibility to induce exon skipping and downregulate MSTN expression in myotubes cultures and DMD mouse models. Furthermore, we combined it with several DMD AONs to look into the possibility of skipping two genes simultaneously (Figure 1).

Bottom Line: Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms.In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients.It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, Leiden, 2300RC, the Netherlands.

ABSTRACT

Background: Myostatin is a potent muscle growth inhibitor that belongs to the Transforming Growth Factor-β (TGF-β) family. Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms. By contrast, Duchenne muscular dystrophy (DMD) is characterized by a loss of muscle fibers and impaired regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients.

Methods: We targeted myostatin exon 2 using antisense oligonucleotides (AON) in healthy and DMD-derived myotubes cultures. We assessed the exon skipping level, transcriptional expression of myostatin and its target genes, and combined myostatin and several dystrophin AONs. These AONs were also applied in the mdx mice models via intramuscular injections.

Results: Myostatin AON induced exon 2 skipping in cell cultures and to a lower extent in the mdx mice. It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression. Furthermore, combination of myostatin and dystrophin AONs induced simultaneous skipping of both genes.

Conclusions: We conclude that two AONs can be used to target two different genes, MSTN and DMD, in a straightforward manner. Targeting multiple ligands of TGF-beta family will be more promising as adjuvant therapies for DMD.

Show MeSH
Related in: MedlinePlus