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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

The expression myogenic regulatory factors MYOG and MYF5 upon exon 2 skipping. Cells were fused and transfected with different concentrations of AON as described in Figure 2. Total RNA was isolated and N6-primed cDNA was subjected to quantitative real-time PCR for MYOG (A) and MYF5 (B). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P < 0.05
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Figure 3: The expression myogenic regulatory factors MYOG and MYF5 upon exon 2 skipping. Cells were fused and transfected with different concentrations of AON as described in Figure 2. Total RNA was isolated and N6-primed cDNA was subjected to quantitative real-time PCR for MYOG (A) and MYF5 (B). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P < 0.05

Mentions: As outlined in Figure 1B, myostatin skipping results in premature stop codon in exon 3 and thus non-functional myostatin protein. Several studies have described that myogenic cells respond to myostatin by down-regulating the expression of key transcriptional regulators of muscle development such as Pax3, Pax7, p21, MyoD, Myf5 and Myog [23-27,45,46], explaining its inhibitory effects in differentiation. Therefore, inhibition of myostatin should lead to an increased expression of these myogenic regulators and thus can be used as a functional readout of the myostatin knockdown. Correspondingly, knockdown of myostatin in differentiated myotubes cultures resulted in a consistent upregulation of MYF5 and MYOG expression (Figure 3A and 3B). The fold increase varied between 3-12 fold and dependent on the myoblast cultures used, which may reflect the different myogenic differentiation potential of the different myoblast cultures.


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)

The expression myogenic regulatory factors MYOG and MYF5 upon exon 2 skipping. Cells were fused and transfected with different concentrations of AON as described in Figure 2. Total RNA was isolated and N6-primed cDNA was subjected to quantitative real-time PCR for MYOG (A) and MYF5 (B). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P < 0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The expression myogenic regulatory factors MYOG and MYF5 upon exon 2 skipping. Cells were fused and transfected with different concentrations of AON as described in Figure 2. Total RNA was isolated and N6-primed cDNA was subjected to quantitative real-time PCR for MYOG (A) and MYF5 (B). Data are means ± SD from 3 to 4 independent experiments. Expression was normalized with GAPDH. Statistical analysis was performed using Student's t-test, using the 500 nM control AON-transfected samples as reference. *P < 0.05
Mentions: As outlined in Figure 1B, myostatin skipping results in premature stop codon in exon 3 and thus non-functional myostatin protein. Several studies have described that myogenic cells respond to myostatin by down-regulating the expression of key transcriptional regulators of muscle development such as Pax3, Pax7, p21, MyoD, Myf5 and Myog [23-27,45,46], explaining its inhibitory effects in differentiation. Therefore, inhibition of myostatin should lead to an increased expression of these myogenic regulators and thus can be used as a functional readout of the myostatin knockdown. Correspondingly, knockdown of myostatin in differentiated myotubes cultures resulted in a consistent upregulation of MYF5 and MYOG expression (Figure 3A and 3B). The fold increase varied between 3-12 fold and dependent on the myoblast cultures used, which may reflect the different myogenic differentiation potential of the different myoblast cultures.

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