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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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Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells. DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. RNA was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by RT-PCR.
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Figure 5: Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells. DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. RNA was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by RT-PCR.

Mentions: To further assess its therapeutic potential, we performed dual exon skipping in the DL589.2 cells, which were derived from a DMD patient with an exon 51-55 deletion. Our previous study has shown that the reading frame can be corrected by an exon 50 skip upon transfection with h50AON1 [7]. As shown in Figure 5, combining this AON with the myostatin AON showed clear targeted skipping of both DMD and MSTN.


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)

Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells. DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. RNA was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Dual exon skipping of myostatin and dystrophin in DL589.2 DMD patient cells. DL589.2 myotubes were transfected with 200 nM of myostatin AON and h50AON1 DMD AON. RNA was isolated two days post-transfection and analyzed for myostatin and dystrophin skips by RT-PCR.
Mentions: To further assess its therapeutic potential, we performed dual exon skipping in the DL589.2 cells, which were derived from a DMD patient with an exon 51-55 deletion. Our previous study has shown that the reading frame can be corrected by an exon 50 skip upon transfection with h50AON1 [7]. As shown in Figure 5, combining this AON with the myostatin AON showed clear targeted skipping of both DMD and MSTN.

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