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Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3

ABSTRACT: First, we compared gene expression and alternative splicing changes between differentially-localized P633L-RBM20, WT-RBM20, and R634Q-RBM20 in iPSC-CMs. Using ICS, we sorted iPSC-CMs with differentially-localized RBM20 based on correlation with nuclear staining. This was followed by RNA-sequencing of the sorted populations. Second, we analysed gene expression and splicing changes in iPSC-CMs overexpressing of WT-, R634Q-, or NLS-tagged R634Q-RBM20 in splice deficient cells carrying the homozygous frameshift mutation (S635FS). Third, we performed RNA sequencing of our HeLa Tet:Cas9 eGFP-RBM20-WT and -R634Q to compare their gene expression to iPSC-CMs. Lastly, we performed two genome-wide CRISPR ICS screens in HeLa cells stably expressing eGFP-RBM20-WT and -R634Q, and TetO-Cas9, to identify genes essential for RBM20 nuclear import.

ORGANISM(S): Homo sapiens

PROVIDER: GSE220833 | GEO | 2023/06/19

REPOSITORIES: GEO

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Project description:Absence of the dystrophin gene in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory medicine, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the efficacy of correction of DMD using the CRISPR/Cas9 system in mitigating the cardiomyopathy phenotype in DMD. To define the effects of CRISPR/Cas9 genome editing on structural, functional and transcriptional dysregulation in DMD-associated cardiomyopathy. We generated induced pluripotent stem cells (iPSCs) from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. Here, we targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD (cDMD) iPSC lines, wherein the DMD open reading frame was restored via reframing (RF) or exon skipping (ES). While DMD cardiomyocytes (CMs) demonstrated morphologic, structural and functional deficits compared to control CMs, CMs from both cDMD lines were similar to control CMs. Bulk RNA-sequencing of DMD CMs showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in cDMD CMs. We then corrected DMD CMs by adenoviral delivery of Cas9/gRNA and showed that postnatal correction of DMD CMs reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts showed reduced transcriptional dysregulation in CMs and fibroblasts in corrected mice compared with DMD mice, consistent with reduced histopathologic changes.We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional and transcriptional dysregulation consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in iPSC-CMs and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.

Project description:Rationale – Absence of the dystrophin gene in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory medicine, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the efficacy of correction of DMD using the CRISPR/Cas9 system in mitigating the cardiomyopathy phenotype in DMD. Objective – To define the effects of CRISPR/Cas9 genome editing on structural, functional and transcriptional dysregulation in DMD-associated cardiomyopathy. Methods and Results – We generated induced pluripotent stem cells (iPSCs) from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. Here, we targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD (cDMD) iPSC lines, wherein the DMD open reading frame was restored via reframing (RF) or exon skipping (ES). While DMD cardiomyocytes (CMs) demonstrated morphologic, structural and functional deficits compared to control CMs, CMs from both cDMD lines were similar to control CMs. Bulk RNA-sequencing of DMD CMs showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in cDMD CMs. We then corrected DMD CMs by adenoviral delivery of Cas9/gRNA and showed that postnatal correction of DMD CMs reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts showed reduced transcriptional dysregulation in CMs and fibroblasts in corrected mice compared with DMD mice, consistent with reduced histopathologic changes. Conclusions – We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional and transcriptional dysregulation consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in iPSC-CMs and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.

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Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3

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