Rationale: Mutations in the cardiac Ryanodine Receptor gene (RYR2) cause dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals exposed to emotions or physical exercise.

Objective: We investigated the efficacy of allele-specific silencing by RNA interference to prevent CPVT phenotypic manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2.

Methods and Results: We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2-R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 (Ryanodine Receptor type 2 protein) function after in vivo delivery by intraperitoneal injection in neonatal and adult RyR2^R4496C/+ (mice heterozygous for the R4496C mutation in the RyR2) heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10, the ratio between wild-type and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2-R4496C mRNA, whereas protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively (1) reduced isoproterenol-induced delayed afterdepolarizations and triggered activity in infected cells, (2) reduced adrenergically mediated ventricular tachycardia in treated mice, (3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and transverse tubules, and (4) attenuated mitochondrial abnormalities.

Conclusions: The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice, suggesting that the reduction of mutant RyR2 may be a novel therapeutic approach for CPVT.

原理：心脏Ryanodine受体基因（ RYR2）的突变会导致儿茶酚胺能性多形性室性心动过速（CPVT）占主导地位，这是在暴露于情绪或体育锻炼的显然健康的个体中突然死亡的主要原因。

目的：我们研究了RNA干扰等位基因特异性沉默在RYR2杂合突变R4496C的优势CPVT小鼠模型携带者中预防CPVT表型表现的功效。

方法和结果：我们开发了一种基于体外mRNA和蛋白质的检测方法，以筛选多个siRNA选择性沉默相关野生型等位基因上的RYR2- R4496C mRNA突变体的能力。对于这些siRNA中性能最高的（siRYR2-U10），我们评估了表达miRYR2-U10的腺相关血清型9病毒载体（AAV9）在通过腹膜内体内递送后纠正RyR2（Ryanodine受体2型蛋白）功能的功效。新生和成年RyR2 ^{R4496C / +}（RyR2中的R4496C突变为杂合小鼠）CPVT杂合小鼠。转录分析表明，用miRYR2-U10处理后，野生型和突变型RYR2的比例mRNA倍增（从1：1到2：1），证实了miRYR2-U10选择性抑制RYR2- R4496C mRNA的能力，而蛋白质定量分析显示，在治疗小鼠的心脏中，总RyR2降低了15％。此外，AAV9-miRYR2-U10有效地（1）减少了异丙肾上腺素引起的延迟后去极化并触发了感染细胞的活性，（2）减少了肾上腺素介导的经治疗的小鼠室性心动过速，（3）交界性肌浆网和横小管的超微结构异常恢复， （4）线粒体异常减弱。

结论：该研究表明，miRYR2-U10等位基因特异性沉默可预防CPVT小鼠危及生命的心律失常，这表明减少RyR2突变体可能是CPVT的一种新型治疗方法。