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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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Administration of myostatin AON in mdx mice. Single dose of 40 μg MSTN AON was injected into the gastrocnemius muscles of mdx mice. Control M23 DMD AON were injected in the contralateral muscles. The animals were sacrificed 4 days after injection. RNA was isolated from three different parts of the muscles relative to the tendon: proximal (P), medial (M) and distal (E). RT-PCR analysis was performed to detect dystrophin (upper) or myostatin (lower) skips (A). Four times consecutive injections were performed with cocktails of AON containing 40 μg of m23 DMD AON and 40 μg of either myostatin or control AON into the gastrocnemius muscles of mdx mice. Injections were varied between contralateral muscles. The mice were sacrificed at 6 hours, 1 day and 2 days after the last injection. RNA was isolated from multiple areas within the muscles and analyzed for dystrophin (upper) or myostatin (lower) skips by RT-PCR (B).
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Figure 6: Administration of myostatin AON in mdx mice. Single dose of 40 μg MSTN AON was injected into the gastrocnemius muscles of mdx mice. Control M23 DMD AON were injected in the contralateral muscles. The animals were sacrificed 4 days after injection. RNA was isolated from three different parts of the muscles relative to the tendon: proximal (P), medial (M) and distal (E). RT-PCR analysis was performed to detect dystrophin (upper) or myostatin (lower) skips (A). Four times consecutive injections were performed with cocktails of AON containing 40 μg of m23 DMD AON and 40 μg of either myostatin or control AON into the gastrocnemius muscles of mdx mice. Injections were varied between contralateral muscles. The mice were sacrificed at 6 hours, 1 day and 2 days after the last injection. RNA was isolated from multiple areas within the muscles and analyzed for dystrophin (upper) or myostatin (lower) skips by RT-PCR (B).

Mentions: We subsequently examined the ability of myostatin AON to induce exon skipping in the mdx mouse model. Myostatin AON was injected at a dose of 40 μg/injection into the gastrocnemius muscle of the mdx mice. DMD AON targeting exon 23 (M23D, previously denoted as M23D(+02-18)[9]) was injected into the contralateral muscle. This AON served as a positive control and was shown by many different groups including ours [9,11] to induce efficient and robust exon skipping. The mice were sacrificed at four days after injection. We classified different sections of the muscles as the proximal (P), medial (M) and distal (D) relative to the tendon and observed heterogenous patterns of Mstn exon 2 skips (Figure 6A). The skipping levels in the proximal and medial parts of the muscles were higher than in the distal, while Dmd skips appeared to be heterogenous throughout the muscles.


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)

Administration of myostatin AON in mdx mice. Single dose of 40 μg MSTN AON was injected into the gastrocnemius muscles of mdx mice. Control M23 DMD AON were injected in the contralateral muscles. The animals were sacrificed 4 days after injection. RNA was isolated from three different parts of the muscles relative to the tendon: proximal (P), medial (M) and distal (E). RT-PCR analysis was performed to detect dystrophin (upper) or myostatin (lower) skips (A). Four times consecutive injections were performed with cocktails of AON containing 40 μg of m23 DMD AON and 40 μg of either myostatin or control AON into the gastrocnemius muscles of mdx mice. Injections were varied between contralateral muscles. The mice were sacrificed at 6 hours, 1 day and 2 days after the last injection. RNA was isolated from multiple areas within the muscles and analyzed for dystrophin (upper) or myostatin (lower) skips by RT-PCR (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Administration of myostatin AON in mdx mice. Single dose of 40 μg MSTN AON was injected into the gastrocnemius muscles of mdx mice. Control M23 DMD AON were injected in the contralateral muscles. The animals were sacrificed 4 days after injection. RNA was isolated from three different parts of the muscles relative to the tendon: proximal (P), medial (M) and distal (E). RT-PCR analysis was performed to detect dystrophin (upper) or myostatin (lower) skips (A). Four times consecutive injections were performed with cocktails of AON containing 40 μg of m23 DMD AON and 40 μg of either myostatin or control AON into the gastrocnemius muscles of mdx mice. Injections were varied between contralateral muscles. The mice were sacrificed at 6 hours, 1 day and 2 days after the last injection. RNA was isolated from multiple areas within the muscles and analyzed for dystrophin (upper) or myostatin (lower) skips by RT-PCR (B).
Mentions: We subsequently examined the ability of myostatin AON to induce exon skipping in the mdx mouse model. Myostatin AON was injected at a dose of 40 μg/injection into the gastrocnemius muscle of the mdx mice. DMD AON targeting exon 23 (M23D, previously denoted as M23D(+02-18)[9]) was injected into the contralateral muscle. This AON served as a positive control and was shown by many different groups including ours [9,11] to induce efficient and robust exon skipping. The mice were sacrificed at four days after injection. We classified different sections of the muscles as the proximal (P), medial (M) and distal (D) relative to the tendon and observed heterogenous patterns of Mstn exon 2 skips (Figure 6A). The skipping levels in the proximal and medial parts of the muscles were higher than in the distal, while Dmd skips appeared to be heterogenous throughout the muscles.

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