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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

AON-mediated skipping of exon 326 in TTN Ser14450fsX4 induced pluripotent stem cell (iPSC)-derived cardiomyocytesRT–PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.RT–PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).
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fig02: AON-mediated skipping of exon 326 in TTN Ser14450fsX4 induced pluripotent stem cell (iPSC)-derived cardiomyocytesRT–PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.RT–PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).

Mentions: We further validated the efficiency of AON1 and AON3 sequences in skipping the human mutated TTN exon 326 by generating virus-free iPSCs from a 62-year-old female affected member of the DCM family carrying the heterozygous TTN Ser14450fsX4 mutation. The presence of the 2-bp AT insertion in exon 326 was confirmed by genomic sequencing (Supplementary Fig S3A). After characterization (Supplementary Figs S3 and S4), two iPSC clones from the DCM patient and an unrelated healthy female were chosen for further studies. Transient transfection of iPSC-derived cardiomyocytes with different doses and combinations of 2OMePS-AONs targeting the human TTN exon 326 and corresponding to the mouse AON1 and AON3 (Supplementary Table S1) resulted in incomplete and unspecific skipping of exon 326 at all concentrations tested, although 2OMePS-AON1 + AON3 promoted the highest amount of correctly skipped transcript in a dose-dependent manner (Fig2A). This was likely due to low transduction efficiency, as demonstrated by transfection of a 5′-fluorescein-labeled 2OMePS AON (Supplementary Fig S5A). In order to enhance nuclear AON delivery in human primary cardiomyocytes, we engineered a lentiviral construct in which the human AON1 and AON3 sequences were embedded in a modified U7 small-nuclear RNA (U7snRNA) followed by an IRES-GFP cassette (U7snRNA-TTNAONs-IRES-GFP). A lentivirus encoding mismatched scrambled AON sequences (U7snRNA-ScrAONs-IRES-GFP) was used as control (Supplementary Fig S5B). U7snRNA is normally involved in histone pre-mRNA 3'-end processing (Galli et al, 1983) and by a small change in the Sm/Lsm protein-binding sites (Stefanovic et al, 1995) can be directed to the spliceosome and used as a shuttle for antisense sequences (Goyenvalle, 2012). After lentiviral infection, ~85% of both control and DCM iPSC-derived cardiomyocytes were GFP positive (GFP+) (Supplementary Fig S5C), and a virtually complete, specific skipping of TTN exon 326 was achieved in both groups exclusively with U7snRNA-TTNAONs-IRES-GFP, as detected by RT–PCR and sequencing (Fig2B and Supplementary Fig S6A). We further confirmed the efficiency of TTN exon 326 skipping at the protein level using mass spectrometry (MS)-based shotgun proteomics (Fig2C and Supplementary Fig S6B). Among ~63,000 detected total peptides, 1,719 corresponded to the human titin protein and 298 mapped to the part encoded by exon 326. Unsupervised hierarchical clustering of titin peptides from control and DCM cardiomyocytes infected with U7snRNA-ScrAONs-IRES-GFP highlighted the presence of a cluster, which was significantly enriched in exon 326 peptides and down-regulated in the diseased cells, as expected (Fig2C). Down-regulation of exon 326 was also detected in DCM cells after treatment with U7snRNA-TTNAONs-IRES-GFP when compared to Scr-AONs, confirming specific skipping of the targeted exon (Fig2C). Similar results were obtained in control cells (Supplementary Fig S7B).


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)

AON-mediated skipping of exon 326 in TTN Ser14450fsX4 induced pluripotent stem cell (iPSC)-derived cardiomyocytesRT–PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.RT–PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).
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fig02: AON-mediated skipping of exon 326 in TTN Ser14450fsX4 induced pluripotent stem cell (iPSC)-derived cardiomyocytesRT–PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.RT–PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).
Mentions: We further validated the efficiency of AON1 and AON3 sequences in skipping the human mutated TTN exon 326 by generating virus-free iPSCs from a 62-year-old female affected member of the DCM family carrying the heterozygous TTN Ser14450fsX4 mutation. The presence of the 2-bp AT insertion in exon 326 was confirmed by genomic sequencing (Supplementary Fig S3A). After characterization (Supplementary Figs S3 and S4), two iPSC clones from the DCM patient and an unrelated healthy female were chosen for further studies. Transient transfection of iPSC-derived cardiomyocytes with different doses and combinations of 2OMePS-AONs targeting the human TTN exon 326 and corresponding to the mouse AON1 and AON3 (Supplementary Table S1) resulted in incomplete and unspecific skipping of exon 326 at all concentrations tested, although 2OMePS-AON1 + AON3 promoted the highest amount of correctly skipped transcript in a dose-dependent manner (Fig2A). This was likely due to low transduction efficiency, as demonstrated by transfection of a 5′-fluorescein-labeled 2OMePS AON (Supplementary Fig S5A). In order to enhance nuclear AON delivery in human primary cardiomyocytes, we engineered a lentiviral construct in which the human AON1 and AON3 sequences were embedded in a modified U7 small-nuclear RNA (U7snRNA) followed by an IRES-GFP cassette (U7snRNA-TTNAONs-IRES-GFP). A lentivirus encoding mismatched scrambled AON sequences (U7snRNA-ScrAONs-IRES-GFP) was used as control (Supplementary Fig S5B). U7snRNA is normally involved in histone pre-mRNA 3'-end processing (Galli et al, 1983) and by a small change in the Sm/Lsm protein-binding sites (Stefanovic et al, 1995) can be directed to the spliceosome and used as a shuttle for antisense sequences (Goyenvalle, 2012). After lentiviral infection, ~85% of both control and DCM iPSC-derived cardiomyocytes were GFP positive (GFP+) (Supplementary Fig S5C), and a virtually complete, specific skipping of TTN exon 326 was achieved in both groups exclusively with U7snRNA-TTNAONs-IRES-GFP, as detected by RT–PCR and sequencing (Fig2B and Supplementary Fig S6A). We further confirmed the efficiency of TTN exon 326 skipping at the protein level using mass spectrometry (MS)-based shotgun proteomics (Fig2C and Supplementary Fig S6B). Among ~63,000 detected total peptides, 1,719 corresponded to the human titin protein and 298 mapped to the part encoded by exon 326. Unsupervised hierarchical clustering of titin peptides from control and DCM cardiomyocytes infected with U7snRNA-ScrAONs-IRES-GFP highlighted the presence of a cluster, which was significantly enriched in exon 326 peptides and down-regulated in the diseased cells, as expected (Fig2C). Down-regulation of exon 326 was also detected in DCM cells after treatment with U7snRNA-TTNAONs-IRES-GFP when compared to Scr-AONs, confirming specific skipping of the targeted exon (Fig2C). Similar results were obtained in control cells (Supplementary Fig S7B).

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