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Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.

Gramlich M, Pane LS, Zhou Q, Chen Z, Murgia M, Schötterl S, Goedel A, Metzger K, Brade T, Parrotta E, Schaller M, Gerull B, Thierfelder L, Aartsma-Rus A, Labeit S, Atherton JJ, McGaughran J, Harvey RP, Sinnecker D, Mann M, Laugwitz KL, Gawaz MP, Moretti A - EMBO Mol Med (2015)

Bottom Line: Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326.Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression.AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University, Tübingen, Germany Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia michael.gramlich@med.uni-tuebingen.de amoretti@med1.med.tum.de.

No MeSH data available.


Related in: MedlinePlus

Efficient skipping of exon 326 in vivo-morpholino-modified antisense oligonucleotide-treated adult Ttn Ser14450fsX4 knock-in miceFluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).RT–PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).
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fig07: Efficient skipping of exon 326 in vivo-morpholino-modified antisense oligonucleotide-treated adult Ttn Ser14450fsX4 knock-in miceFluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).RT–PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).

Mentions: To assess whether the functional improvement was associated with exon skipping, animals were sacrificed at the end of the 2-week treatment and molecular analyses were performed in ventricular tissue (Fig1). Fluorescence in situ hybridization (FISH) with a specific probe complementary to the mTtnAON sequence (Ttn-A probe) demonstrated an almost complete penetrance of the vivo-morpholino into the heart of the vPMO-mTtnAON-treated HET animals, while no signal was detected in HET mice that received the vPMO-mScrAON (Fig7A and Supplementary Fig S9D). RT–PCR and direct sequencing of cardiac cDNA demonstrated that vPMO-mTtnAON treatment restored the Ttn reading frame, although the resulted transcript was shorter than expected, including the first 189 bp of exon 326 and lacking the first 267 bp of exon 327 (Fig7B). Finally, evaluation of titin protein by MS-based proteomic analysis revealed a down-regulation of peptides mapping to mouse exon 326 and an up-regulation of peptides downstream of exon 326 (that we refer as ‘C-terminus') in ventricular tissue of HET animals treated with vPMO-mTtnAON compared to vPMO-mScrAON (Fig7C). By calculating the percentage change of the relative intensity of ‘C-terminus' peptides to all titin peptides, we estimated an ~8% skipping efficiency of the mutated 326 exon after the application of vPMO-mTtnAON (see Supplementary Information for details). These results are in accordance with data in dystrophic mdx mice reporting a ~5–10% dystrophin induction in heart muscle after intravenous injection of 6–300 mg/kg morpholinoE23 (Wu et al,2010).


Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.

Gramlich M, Pane LS, Zhou Q, Chen Z, Murgia M, Schötterl S, Goedel A, Metzger K, Brade T, Parrotta E, Schaller M, Gerull B, Thierfelder L, Aartsma-Rus A, Labeit S, Atherton JJ, McGaughran J, Harvey RP, Sinnecker D, Mann M, Laugwitz KL, Gawaz MP, Moretti A - EMBO Mol Med (2015)

Efficient skipping of exon 326 in vivo-morpholino-modified antisense oligonucleotide-treated adult Ttn Ser14450fsX4 knock-in miceFluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).RT–PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492817&req=5

fig07: Efficient skipping of exon 326 in vivo-morpholino-modified antisense oligonucleotide-treated adult Ttn Ser14450fsX4 knock-in miceFluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).RT–PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).
Mentions: To assess whether the functional improvement was associated with exon skipping, animals were sacrificed at the end of the 2-week treatment and molecular analyses were performed in ventricular tissue (Fig1). Fluorescence in situ hybridization (FISH) with a specific probe complementary to the mTtnAON sequence (Ttn-A probe) demonstrated an almost complete penetrance of the vivo-morpholino into the heart of the vPMO-mTtnAON-treated HET animals, while no signal was detected in HET mice that received the vPMO-mScrAON (Fig7A and Supplementary Fig S9D). RT–PCR and direct sequencing of cardiac cDNA demonstrated that vPMO-mTtnAON treatment restored the Ttn reading frame, although the resulted transcript was shorter than expected, including the first 189 bp of exon 326 and lacking the first 267 bp of exon 327 (Fig7B). Finally, evaluation of titin protein by MS-based proteomic analysis revealed a down-regulation of peptides mapping to mouse exon 326 and an up-regulation of peptides downstream of exon 326 (that we refer as ‘C-terminus') in ventricular tissue of HET animals treated with vPMO-mTtnAON compared to vPMO-mScrAON (Fig7C). By calculating the percentage change of the relative intensity of ‘C-terminus' peptides to all titin peptides, we estimated an ~8% skipping efficiency of the mutated 326 exon after the application of vPMO-mTtnAON (see Supplementary Information for details). These results are in accordance with data in dystrophic mdx mice reporting a ~5–10% dystrophin induction in heart muscle after intravenous injection of 6–300 mg/kg morpholinoE23 (Wu et al,2010).

Bottom Line: Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326.Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression.AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University, Tübingen, Germany Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia michael.gramlich@med.uni-tuebingen.de amoretti@med1.med.tum.de.

No MeSH data available.


Related in: MedlinePlus