Dave Jaydev, Raad Nour, Mittal Nishka, Zhang Lu, Fargnoli Anthony, Oh Jae Gyun, Savoia Maria Elisabetta, Hansen Jens, Fava Marika, Yin Xiaoke, Theofilatos Konstantinos, Ceholski Delaine, Kohlbrenner Erik, Jeong Dongtak, Wills Lauren, Nonnenmacher Mathieu, Haghighi Kobra, Costa Kevin D, Turnbull Irene C, Mayr Manuel, Cai Chen-Leng, Kranias Evangelia G, Akar Fadi G, Hajjar Roger J, Stillitano Francesca
Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Department of Pediatrics, Riley Heart Research Center, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
Cardiovasc Res. 2022 Dec 9;118(15):3140-3150. doi: 10.1093/cvr/cvac021.
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-9 (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del.
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 polymerase chain reaction 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).
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.
磷酸受磷蛋白（PLN）基因中的一种突变导致第14位精氨酸缺失（R14del），与恶性心律失常和心室扩张有关。识别具有易损心肌的症状前携带者至关重要，因为心律失常可导致心源性猝死，尤其是在携带PLN-R14del突变的年轻人中。本研究旨在评估使用CRISPR/Cas9和心肌嗜性腺相关病毒9型（AAV9）进行体内基因组编辑改善表达人PLN-R14del的年轻成年小鼠心脏功能的效率和效果。
生成杂合状态下表达人野生型（hPLN-WT）或突变型（hPLN-R14del）PLN的人源化小鼠，模拟人类携带者。12周龄时的心脏磁共振成像显示，与野生型小鼠相比，突变型小鼠出现双心室扩张且每搏输出量增加，射血分数或心输出量无缺陷。用异丙肾上腺素和快速起搏对离体心脏进行刺激发现，相对于hPLN-WT，hPLN-R14del发生持续性室性心动过速（VT）的倾向更高。具体而言，VT阈值显著降低（hPLN-R14del为20.3±1.2Hz，野生型为25.7±1.3Hz，P<0.01），反映出心律失常负担更高。为了使R14del等位基因失活，给小鼠尾静脉注射AAV9.CRISPR/Cas9/gRNA或AAV9空衣壳（对照）。通过液滴数字聚合酶链反应和基于NGS的扩增子测序评估CRISPR-Cas9效率。与对照组相比，体内基因编辑显著降低了hPLN-R14del CRISPR处理小鼠的舒张末期容积和每搏输出量。VT易感性也降低了，因为相对于对照组，VT阈值显著升高（30.9±2.3Hz对21.3±1.5Hz；P<0.01）。
本研究首次表明，AAV9-CRISPR/Cas9破坏hPLN-R14del等位基因可改善人源化PLN-R14del小鼠的心脏功能并降低VT易感性，为治疗性抑制PLN-R14del疾病人类患者致心律失常表型的可转化方法提供了临床前证据。
