Genome Editing Treatment for Catecholaminergic Polymorphic Ventricular Tachycardia

在治疗儿茶酚胺能多形性室性心动过速（CPVT）方面存在根本性差距。 CPVT的药物治疗对导致猝死的疾病部分有效。更全面 需要了解新的基因编辑方法，以开发安全，非手术和有效的 针对CPVT的根本原因的治疗。在大约60%的CPVT患者中， RYR 2通过心肌细胞中的异常钙处理引起心律失常。实验表明，使用 CRISPR/Cas9敲低RYR 2突变等位基因通过以下方式有效预防室性心动过速（VT）： 减少功能失调的RyR 2的表达。本申请的总体目标是改善治疗， CPVT患者申请人生成的初步数据显示，直接针对疾病- 在CPVT小鼠模型中用传统CRISPR/Cas9疗法引起突变可预防起搏诱导的VT 但降低RyR 2的总表达。这项提议的核心假设是， CRISPR/Cas9基因编辑可以通过特异性纠正致病突变位点来治疗CPVT， RyR 2表达，同时保持总RyR 2表达，使Ca 2+处理正常化，并降低RyR 2表达。 易患VT。这项研究的基本原理是，了解有效性和特异性 基因编辑在纠正心脏突变中的应用可能会导致安全的新方法来治疗遗传病。 心脏疾病该假设将通过以下具体目的进行检验：1）检验治疗潜力 2）在CPVT的iPSC-CM临床前模型中测试人RYR 2突变校正的治疗效果， CPVT小鼠模型中小鼠RYR 2突变校正的潜力。为了确定目标1，我们提供 CRISPR/Cas9引物编辑载体IPSC，衍生CM，并通过共聚焦和光测量功能 IPSC和3D心脏类器官的薄片成像。为了确定目标2，我们将使用Lenti-CRISPR/Cas9引物， 编辑载体以治疗CPVT小鼠模型并通过以下方式测量长期心脏功能： 超声心动图、EKG、程控电刺激和分子分析。本研究是 重要的是，它将推进RYR 2突变的基因编辑校正，作为一种安全有效的方法， CPVT的治疗

There is a fundamental gap in treatment for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Pharmacological treatment of CPVT is partially effective for a disease that causes sudden death. A fuller understanding of novel gene editing approaches is needed to develop safe, non-surgical, and effective therapies that target the underlying causes of CPVT. In approximately 60% of CPVT patients, mutations in RYR2 cause arrhythmia through abnormal calcium handling in cardiomyocytes. The lab has shown that using CRISPR/Cas9 to knockdown the RYR2 mutant allele is effective in preventing ventricular tachycardia(VT) by decreasing expression of dysfunctional RyR2. The overall objective of this application is to improve therapy for patients with CPVT. Preliminary data generated by the applicant showed that directly targeting a disease- causing mutation with traditional CRISPR/Cas9 therapy in a CPVT mouse model prevented pacing induced VT but reduced total expression of RyR2. The central hypothesis of this proposal is that novel methods of CRISPR/Cas9 gene editing can treat CPVT by specifically correcting causative mutation sites, reducing mutant RyR2 expression while preserving total RyR2 expression, normalizing Ca2+ handling, and decreasing susceptibility to VT. The rationale for this research is that an understanding of the effectiveness and specificity of gene editing in the correcting mutations in the heart may lead to safe novel approaches to treat genetic cardiac disorders. The hypothesis will be tested with the following specific aims 1) test the therapeutic potential of human RYR2 mutation correction in an iPSC-CM preclinical model of CPVT and 2) test the therapeutic potential of mouse RYR2 mutation correction in a mouse model of CPVT. To determine aim 1, we deliver CRISPR/Cas9 prime editing vectors to IPSC, derive CM, and measure function through confocal and light sheet imaging of IPSC and 3D cardiac organoids. To determine aim 2, we will use Lenti-CRISPR/Cas9 prime editing vectors to treat mouse models of CPVT and measure long-term cardiac function through echocardiography, EKG, programmed electrical stimulation, and molecular analysis. This research is significant in that it will advance gene editing correction of RYR2 mutations as a safe and effective method for the treatment of CPVT.