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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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Myostatin exon 2 skip in several myotubes cultures. Human primary control (KM109) and DMD patient derived- (DL589.2) myoblasts were differentiated for 7 days before transfection with MSTN AON. Immortalized control (7304.1) myoblasts were differentiated for 2-3 days. A non-targeting, fluorescently-labeled AONs were transfected as control. Fluorescent nuclei were observed three hours post-transfection (A). RNA was isolated 2 days post-transfection. cDNA was synthesized using random hexamer (N6) primers and subjected for PCR using primers in exon 1 and 3 (B). Note the inverse dose-dependent skips in KM109 samples. Skip fragment was confirmed by sequencing analysis (C). Quantitative real-time PCR was performed using primers in MSTN exon 1 and 2, thereby depicting the expression of remaining full length or non-skipped transcript (D). 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 2: Myostatin exon 2 skip in several myotubes cultures. Human primary control (KM109) and DMD patient derived- (DL589.2) myoblasts were differentiated for 7 days before transfection with MSTN AON. Immortalized control (7304.1) myoblasts were differentiated for 2-3 days. A non-targeting, fluorescently-labeled AONs were transfected as control. Fluorescent nuclei were observed three hours post-transfection (A). RNA was isolated 2 days post-transfection. cDNA was synthesized using random hexamer (N6) primers and subjected for PCR using primers in exon 1 and 3 (B). Note the inverse dose-dependent skips in KM109 samples. Skip fragment was confirmed by sequencing analysis (C). Quantitative real-time PCR was performed using primers in MSTN exon 1 and 2, thereby depicting the expression of remaining full length or non-skipped transcript (D). 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: To examine the feasibility to induce myostatin exon 2 skipping, different concentrations of each AON were transfected into the myotube cultures using the cationic polymer polyethylenimine (PEI). More than 80% of the cells showed specific nuclear uptake upon transfection with 5'-fluorescein (FAM)-labeled control AON (Figure 2A). RT-PCR performed two days post transfection (Figure 2B) and subsequent sequencing analysis (Figure 2C) showed the exclusion of exon 2 from the myostatin transcript in the myostatin AON-transfected cells, resulting in a premature stop codon formation. This internally truncated fragment was not observed in any of the non-transfected and control AON-transfected myotubes. One myostatin AON, namely AON1, gave the most consistent and highest skipping efficiency [Additional file 1]. Thus we further used the AON1 (addressed as myostatin AON from now on) and confirmed its exon skipping ability in human and to a lower extent in mouse cells models, using its perfect complementary to the human and mouse MSTN sequences (Figure 2B and not shown).


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)

Myostatin exon 2 skip in several myotubes cultures. Human primary control (KM109) and DMD patient derived- (DL589.2) myoblasts were differentiated for 7 days before transfection with MSTN AON. Immortalized control (7304.1) myoblasts were differentiated for 2-3 days. A non-targeting, fluorescently-labeled AONs were transfected as control. Fluorescent nuclei were observed three hours post-transfection (A). RNA was isolated 2 days post-transfection. cDNA was synthesized using random hexamer (N6) primers and subjected for PCR using primers in exon 1 and 3 (B). Note the inverse dose-dependent skips in KM109 samples. Skip fragment was confirmed by sequencing analysis (C). Quantitative real-time PCR was performed using primers in MSTN exon 1 and 2, thereby depicting the expression of remaining full length or non-skipped transcript (D). 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 2: Myostatin exon 2 skip in several myotubes cultures. Human primary control (KM109) and DMD patient derived- (DL589.2) myoblasts were differentiated for 7 days before transfection with MSTN AON. Immortalized control (7304.1) myoblasts were differentiated for 2-3 days. A non-targeting, fluorescently-labeled AONs were transfected as control. Fluorescent nuclei were observed three hours post-transfection (A). RNA was isolated 2 days post-transfection. cDNA was synthesized using random hexamer (N6) primers and subjected for PCR using primers in exon 1 and 3 (B). Note the inverse dose-dependent skips in KM109 samples. Skip fragment was confirmed by sequencing analysis (C). Quantitative real-time PCR was performed using primers in MSTN exon 1 and 2, thereby depicting the expression of remaining full length or non-skipped transcript (D). 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: To examine the feasibility to induce myostatin exon 2 skipping, different concentrations of each AON were transfected into the myotube cultures using the cationic polymer polyethylenimine (PEI). More than 80% of the cells showed specific nuclear uptake upon transfection with 5'-fluorescein (FAM)-labeled control AON (Figure 2A). RT-PCR performed two days post transfection (Figure 2B) and subsequent sequencing analysis (Figure 2C) showed the exclusion of exon 2 from the myostatin transcript in the myostatin AON-transfected cells, resulting in a premature stop codon formation. This internally truncated fragment was not observed in any of the non-transfected and control AON-transfected myotubes. One myostatin AON, namely AON1, gave the most consistent and highest skipping efficiency [Additional file 1]. Thus we further used the AON1 (addressed as myostatin AON from now on) and confirmed its exon skipping ability in human and to a lower extent in mouse cells models, using its perfect complementary to the human and mouse MSTN sequences (Figure 2B and not shown).

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