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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 control cells. KM109 (A) and 7304-1 (B) myotubes were transfected with 200 nM of myostatin AON and AON targeting different DMD exons, namely exon 8, 44 and 54. The AONs were premixed (boxed) before complexing with the transfection reagent, or directly complexed (not boxed). RNA was isolated two days post-transfection and analyzed for myostatin or dystrophin skips by RT-PCR.
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Figure 4: Dual exon skipping of myostatin and dystrophin in control cells. KM109 (A) and 7304-1 (B) myotubes were transfected with 200 nM of myostatin AON and AON targeting different DMD exons, namely exon 8, 44 and 54. The AONs were premixed (boxed) before complexing with the transfection reagent, or directly complexed (not boxed). RNA was isolated two days post-transfection and analyzed for myostatin or dystrophin skips by RT-PCR.

Mentions: Our ultimate goal was to use AONs to simultaneously reframe the mutation in the DMD gene and to downregulate MSTN expression in order to correct the primary genetic defect as well as to enhance muscle regeneration. Therefore, we combined the myostatin AON with several DMD AONs used in the previous studies [6,7,47]. We specifically applied DMD AONs covering the hotspot mutated regions in DMD gene, namely h8AON3, h44AON1, h51AON1 and h54AON1 (Table 1) in control myotubes cultures (primary (KM109) or immortalized myoblasts (7304-1)). Myotubes were transfected with either myostatin or DMD AONs or a mix of both. As shown in Figure 4, MSTN exon skip was observed in all myostatin AON-transfected samples, regardless of the presence of DMD AON. Vice versa, all DMD AON-transfected myotubes showed the exon-specific dystrophin skips described before [6,7,47], regardless of the presence of MSTN AON. To investigate the possibility that the two AONs hybridized to each other rather than to their intended target, we varied the transfection conditions by mixing the AON before complexing with PEI, or preparing the AON-PEI complexes separately. However, no difference in their efficiency to induce skipping was found. We observed skipping with the total AON concentration of 200 nM (Figure 4) as well as 100 nM (not shown). These results demonstrated the feasibility of simultaneous antisense-mediated skipping of exons of two different genes.


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 control cells. KM109 (A) and 7304-1 (B) myotubes were transfected with 200 nM of myostatin AON and AON targeting different DMD exons, namely exon 8, 44 and 54. The AONs were premixed (boxed) before complexing with the transfection reagent, or directly complexed (not boxed). RNA was isolated two days post-transfection and analyzed for myostatin or dystrophin skips by RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Dual exon skipping of myostatin and dystrophin in control cells. KM109 (A) and 7304-1 (B) myotubes were transfected with 200 nM of myostatin AON and AON targeting different DMD exons, namely exon 8, 44 and 54. The AONs were premixed (boxed) before complexing with the transfection reagent, or directly complexed (not boxed). RNA was isolated two days post-transfection and analyzed for myostatin or dystrophin skips by RT-PCR.
Mentions: Our ultimate goal was to use AONs to simultaneously reframe the mutation in the DMD gene and to downregulate MSTN expression in order to correct the primary genetic defect as well as to enhance muscle regeneration. Therefore, we combined the myostatin AON with several DMD AONs used in the previous studies [6,7,47]. We specifically applied DMD AONs covering the hotspot mutated regions in DMD gene, namely h8AON3, h44AON1, h51AON1 and h54AON1 (Table 1) in control myotubes cultures (primary (KM109) or immortalized myoblasts (7304-1)). Myotubes were transfected with either myostatin or DMD AONs or a mix of both. As shown in Figure 4, MSTN exon skip was observed in all myostatin AON-transfected samples, regardless of the presence of DMD AON. Vice versa, all DMD AON-transfected myotubes showed the exon-specific dystrophin skips described before [6,7,47], regardless of the presence of MSTN AON. To investigate the possibility that the two AONs hybridized to each other rather than to their intended target, we varied the transfection conditions by mixing the AON before complexing with PEI, or preparing the AON-PEI complexes separately. However, no difference in their efficiency to induce skipping was found. We observed skipping with the total AON concentration of 200 nM (Figure 4) as well as 100 nM (not shown). These results demonstrated the feasibility of simultaneous antisense-mediated skipping of exons of two different genes.

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