Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact

Abstract

Aims A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. Methods and Results Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3±1.2 Hz in hPLN-R14del vs. 25.7±1.3 Hz in WT, p < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital PCR and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9±2.3 Hz vs. 21.3±1.5 Hz; p < 0.01). Conclusions This study is the first to show that disruption of hPLN-R14del allele by AAV9- CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease. Translational Perspective The phospholamban R14del mutation causes dilated and arrhythmogenic cardiomyopathies, with increased risk of malignant ventricular arrhythmias in young adult carriers. With few available therapeutic options, heart transplantation is often the ultimate treatment. This study presents the first humanized mouse model of PLN-R14del disease, reveals the ability to detect abnormal cardiac function and increased arrhythmogenic vulnerability in pre-symptomatic hPLN-R14del mice, and demonstrates that allele-specific disruption of R14del using in vivo AAV9/CRISPR-Cas9 reverses the disease phenotype. This preclinical study offers promising translatable approaches to detect and therapeutically suppress the arrhythmogenic phenotype in patients with PLN-R14del disease and potentially other inherited cardiomyopathies.

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca2+ Handling and Arrhythmogenesis

“…Based on initial findings from PLN-R14del overexpression in a heterologous cell culture system as well as in the mouse heart, PLN-R14del was proposed to exert super-inhibitory effects on SERCA2a activity, leading to cardiac remodeling and early death [5]. Additional evidence has recently emerged from numerous studies on human patients and/or patient-derived cardiomyocytes (iPSC-CMs), and various PLN-R14del animal models [12][13][14][15][16][17][18]. These studies have revealed multiple defects associated with the PLN-R14del mutation, including impaired Ca 2+ homeostasis [12][13][14]16,18], electrical remodeling [19], unfolded protein response (UPR) activation [20], and protein aggregation [15,21,22].…”

“…Additional evidence has recently emerged from numerous studies on human patients and/or patient-derived cardiomyocytes (iPSC-CMs), and various PLN-R14del animal models [12][13][14][15][16][17][18]. These studies have revealed multiple defects associated with the PLN-R14del mutation, including impaired Ca 2+ homeostasis [12][13][14]16,18], electrical remodeling [19], unfolded protein response (UPR) activation [20], and protein aggregation [15,21,22].…”

Computational design of custom therapeutic cells to correct failing human cardiomyocytes

“…For example, herein we assume all cardiomyocytes will need to be sufficiently restored, requiring an abundant number of therapeutic cells to be delivered (2-5 custom cells per cardiomyocyte). However, recent gene therapy studies have shown that correcting only a small percentage of cardiomyocytes can improve muscle function ( Long et al, 2016 ; Nelson et al, 2016 ; Dave et al, 2022 ). Further in vitro and in vivo validation studies are therefore warranted to explore the cell coupling efficiency required to achieve a therapeutically beneficial restoration effect.…”

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in Cardiovascular Disease: A Comprehensive Clinical Review on Dilated Cardiomyopathy

“…Dave et al (2022) aimed to assess the therapeutic effects of CRISPR/Cas9-mediated genome editing on cardiac function in mice carrying a mutation in the PLN9 gene [ 69 ]. The authors employed a cardiotropic AAV9 vector to deliver CRISPR/Cas9 and guide RNA (gRNA) effectively disrupting the gene and evaluated the efficacy of the gene editing using droplet digital polymerase chain reaction and next-generation sequencing (NGS)-based amplicon sequencing [ 69 ]. The results indicated that mutant mice had bi-ventricular dilation and increased stroke volume compared to wild-type mice, with a higher propensity for sustained ventricular tachycardia [ 69 ].…”

“…The authors employed a cardiotropic AAV9 vector to deliver CRISPR/Cas9 and guide RNA (gRNA) effectively disrupting the gene and evaluated the efficacy of the gene editing using droplet digital polymerase chain reaction and next-generation sequencing (NGS)-based amplicon sequencing [ 69 ]. The results indicated that mutant mice had bi-ventricular dilation and increased stroke volume compared to wild-type mice, with a higher propensity for sustained ventricular tachycardia [ 69 ]. This is followed by in vivo gene editing to correct the induced mutation, which significantly reduced end-diastolic and stroke volumes and susceptibility to ventricular tachycardia [ 69 ].…”