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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.

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Related in: MedlinePlus

Exon skipping-based rescue of the DCM phenotype in Ttn Ser14450fsX4 knock-in embryosA Experimental design.B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).Data information: Statistical difference was tested using the two-sided Student's t-test (**P = 0.007 and *P = 0.01 for fractional area change and **P = 0.009 for filament width differences). Data represent mean values ± SEM. Scale bars, 0.2 μm.
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fig05: Exon skipping-based rescue of the DCM phenotype in Ttn Ser14450fsX4 knock-in embryosA Experimental design.B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).Data information: Statistical difference was tested using the two-sided Student's t-test (**P = 0.007 and *P = 0.01 for fractional area change and **P = 0.009 for filament width differences). Data represent mean values ± SEM. Scale bars, 0.2 μm.

Mentions: We further assessed the efficacy and impact of reframing Ttn transcripts by AON-mediated skipping in the heart muscle and used the mouse DCM model based on the human TTN exon 326 A-band truncating mutation (Gramlich et al, 2009). We generated U7snRNA-AONs-IRES-GFP lentiviral constructs encoding the mouse specific AON1/3 (U7snRNA-mTtnAONs-IRES-GFP) or scrambled AON (U7snRNA-mSrcAONs-IRES-GFP) sequences. We first analyzed knock-in mouse embryos, which were collected at day 8.5 and cultured for 24 h after lentiviral infection or transient transfection of mouse 2OMePS oligos—AON1/3 (mTtnAON) or scrambled AONs (mScrAON)—in order to compare the efficacy of the two delivery methods (Fig5A). Wild-type (WT) untreated hearts displayed proper formation of sarcomeres with clearly distinguishable Z-disks (Fig5B) and beat normally (Fig5C). In contrast, homozygous Ttn-mutant myocardium of the same stage showed no striations (Fig5B), indicative of impaired sarcomerogenesis, and consequently did not develop contractile function (Fig5C). Mutants treated with mTtnAONs exhibited rescued sarcomere assembly and Z-disk formation (Fig5B) and a significant increase in contractile function when compared to untreated or mScrAON-treated homozygous littermates (Fig5C). Moreover, mTtnAON application restored normal filament width, as detected by electron microscopy (Fig5D and E). Beneficial effects after mTtnAON treatment were similar in both 2OMePS- and lentivirus-based systems (Fig5B and C), suggesting a comparable efficacy of the two methods in transducing the myocardium ex vivo.


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)

Exon skipping-based rescue of the DCM phenotype in Ttn Ser14450fsX4 knock-in embryosA Experimental design.B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).Data information: Statistical difference was tested using the two-sided Student's t-test (**P = 0.007 and *P = 0.01 for fractional area change and **P = 0.009 for filament width differences). Data represent mean values ± SEM. Scale bars, 0.2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Exon skipping-based rescue of the DCM phenotype in Ttn Ser14450fsX4 knock-in embryosA Experimental design.B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).Data information: Statistical difference was tested using the two-sided Student's t-test (**P = 0.007 and *P = 0.01 for fractional area change and **P = 0.009 for filament width differences). Data represent mean values ± SEM. Scale bars, 0.2 μm.
Mentions: We further assessed the efficacy and impact of reframing Ttn transcripts by AON-mediated skipping in the heart muscle and used the mouse DCM model based on the human TTN exon 326 A-band truncating mutation (Gramlich et al, 2009). We generated U7snRNA-AONs-IRES-GFP lentiviral constructs encoding the mouse specific AON1/3 (U7snRNA-mTtnAONs-IRES-GFP) or scrambled AON (U7snRNA-mSrcAONs-IRES-GFP) sequences. We first analyzed knock-in mouse embryos, which were collected at day 8.5 and cultured for 24 h after lentiviral infection or transient transfection of mouse 2OMePS oligos—AON1/3 (mTtnAON) or scrambled AONs (mScrAON)—in order to compare the efficacy of the two delivery methods (Fig5A). Wild-type (WT) untreated hearts displayed proper formation of sarcomeres with clearly distinguishable Z-disks (Fig5B) and beat normally (Fig5C). In contrast, homozygous Ttn-mutant myocardium of the same stage showed no striations (Fig5B), indicative of impaired sarcomerogenesis, and consequently did not develop contractile function (Fig5C). Mutants treated with mTtnAONs exhibited rescued sarcomere assembly and Z-disk formation (Fig5B) and a significant increase in contractile function when compared to untreated or mScrAON-treated homozygous littermates (Fig5C). Moreover, mTtnAON application restored normal filament width, as detected by electron microscopy (Fig5D and E). Beneficial effects after mTtnAON treatment were similar in both 2OMePS- and lentivirus-based systems (Fig5B and C), suggesting a comparable efficacy of the two methods in transducing the myocardium ex vivo.

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