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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

Prevention of DCM phenotype development in adult Ttn Ser14450fsX4 knock-in mice by injection of vivo-morpholino-modified antisense oligonucleotidesExperimental design.Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.
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fig06: Prevention of DCM phenotype development in adult Ttn Ser14450fsX4 knock-in mice by injection of vivo-morpholino-modified antisense oligonucleotidesExperimental design.Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.

Mentions: Although used in several of the preclinical and early clinical trials for DMD, the 2OMePS oligos are reported to have low effectiveness in targeting cardiac muscle in vivo (Heemskerk et al, 2009; Betts & Wood, 2013). Indeed, in a preliminary screening for AON chemistry that allows skipping of Ttn exon 326 in the heart in vivo, systemic infusion of the mouse 2OMePS-AON 1 and 3 did not produce any skipping of cardiac Ttn in HET animals, even at the highest doses tested (Supplementary Fig S9A), while local skeletal muscle injection of the same 2OMePS AONs led to correct skipping of such exon in the skeletal muscle transcript (Supplementary Fig S9A). Therefore, we chose to use vivo-morpholino-modified AONs (vPMO-AONs, see Materials and Methods), which have been described to better penetrate heart tissue (Mendell et al, 2013). Intraperitoneal (I.P.) injection of 6 mg/kg body weight of mouse vPMO-AON1 and 3 allowed skipping of cardiac Ttn exon 326 in HET mice, but in an incomplete and unspecific manner (Supplementary Fig S9B). However, extension of the v-PMO-AON1 sequence from a 23mer to a 28mer markedly improved AON efficiency and specificity (Supplementary Fig S9C). Therefore, the 28-mer vPMO-AON1 (vPMO-mTtnAON) and its correspondent scrambled sequence (vPMO-mScrAON) were further used in our in vivo studies. Figure6A illustrates our experimental design. Heterozygous Ttn knock-in mice did not show any obvious cardiac phenotype under sedentary conditions. However, when exposed to cardiac stress they develop features of DCM (Gramlich et al, 2009). Thus, we implanted osmotic minipumps delivering Ang II (1.4 mg/kg) in WT (n = 6) and HET animals (n = 30). One subgroup of HET mice was intraperitoneally injected with 6 mg/kg body weight of vPMO-mTtnAON (n = 14) at day 0 and day 7. Another subgroup received the same dose of a mismatched vPMO-mScrAON (n = 10) or no oligos (n = 6) (Fig6A). Echocardiographic analysis of systolic and diastolic function revealed that all subgroups developed a similar hypertrophic response after 1 week of Ang II infusion, with decreased ventricular end-diastolic diameters (LVEDD) and increased left ventricular ejection fraction (LV-EF) and wall thicknesses (IVSd and PWd) (Fig6B). Consistent with our previous findings (Gramlich et al, 2009), after 2 weeks of Ang II HET animals that were injected with saline or vPMO-mScrAON displayed a DCM-like phenotype characterized by a reduction in LV-EF, IVSd, and PWd and by an increase in LVEDD (Fig6B). In contrast, vPMO-mTtnAON-treated HET mutants did not develop DCM and showed a response similar to WT animals with continued hypertrophy (Fig6B). Furthermore, vPMO-mTtnAON injections in HET animals reversed the development of interstitial fibrosis that typically occurs in these mice after a 2-week treatment with Ang II (Fig6C). At the end of each experiment, the animals were carefully analyzed for organ damage (e.g., liver failure). We could not detect any signs of toxicity in vPMO-treated mice.


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)

Prevention of DCM phenotype development in adult Ttn Ser14450fsX4 knock-in mice by injection of vivo-morpholino-modified antisense oligonucleotidesExperimental design.Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig06: Prevention of DCM phenotype development in adult Ttn Ser14450fsX4 knock-in mice by injection of vivo-morpholino-modified antisense oligonucleotidesExperimental design.Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.
Mentions: Although used in several of the preclinical and early clinical trials for DMD, the 2OMePS oligos are reported to have low effectiveness in targeting cardiac muscle in vivo (Heemskerk et al, 2009; Betts & Wood, 2013). Indeed, in a preliminary screening for AON chemistry that allows skipping of Ttn exon 326 in the heart in vivo, systemic infusion of the mouse 2OMePS-AON 1 and 3 did not produce any skipping of cardiac Ttn in HET animals, even at the highest doses tested (Supplementary Fig S9A), while local skeletal muscle injection of the same 2OMePS AONs led to correct skipping of such exon in the skeletal muscle transcript (Supplementary Fig S9A). Therefore, we chose to use vivo-morpholino-modified AONs (vPMO-AONs, see Materials and Methods), which have been described to better penetrate heart tissue (Mendell et al, 2013). Intraperitoneal (I.P.) injection of 6 mg/kg body weight of mouse vPMO-AON1 and 3 allowed skipping of cardiac Ttn exon 326 in HET mice, but in an incomplete and unspecific manner (Supplementary Fig S9B). However, extension of the v-PMO-AON1 sequence from a 23mer to a 28mer markedly improved AON efficiency and specificity (Supplementary Fig S9C). Therefore, the 28-mer vPMO-AON1 (vPMO-mTtnAON) and its correspondent scrambled sequence (vPMO-mScrAON) were further used in our in vivo studies. Figure6A illustrates our experimental design. Heterozygous Ttn knock-in mice did not show any obvious cardiac phenotype under sedentary conditions. However, when exposed to cardiac stress they develop features of DCM (Gramlich et al, 2009). Thus, we implanted osmotic minipumps delivering Ang II (1.4 mg/kg) in WT (n = 6) and HET animals (n = 30). One subgroup of HET mice was intraperitoneally injected with 6 mg/kg body weight of vPMO-mTtnAON (n = 14) at day 0 and day 7. Another subgroup received the same dose of a mismatched vPMO-mScrAON (n = 10) or no oligos (n = 6) (Fig6A). Echocardiographic analysis of systolic and diastolic function revealed that all subgroups developed a similar hypertrophic response after 1 week of Ang II infusion, with decreased ventricular end-diastolic diameters (LVEDD) and increased left ventricular ejection fraction (LV-EF) and wall thicknesses (IVSd and PWd) (Fig6B). Consistent with our previous findings (Gramlich et al, 2009), after 2 weeks of Ang II HET animals that were injected with saline or vPMO-mScrAON displayed a DCM-like phenotype characterized by a reduction in LV-EF, IVSd, and PWd and by an increase in LVEDD (Fig6B). In contrast, vPMO-mTtnAON-treated HET mutants did not develop DCM and showed a response similar to WT animals with continued hypertrophy (Fig6B). Furthermore, vPMO-mTtnAON injections in HET animals reversed the development of interstitial fibrosis that typically occurs in these mice after a 2-week treatment with Ang II (Fig6C). At the end of each experiment, the animals were carefully analyzed for organ damage (e.g., liver failure). We could not detect any signs of toxicity in vPMO-treated mice.

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