Genome Editing Treatment for Catecholaminergic Polymorphic Ventricular Tachycardia

儿茶酚胺能多形性室性心动过速(CPVT)的治疗存在一个根本性的缺口。 CPVT的药物治疗对导致猝死的疾病部分有效。更饱满的人 为了开发安全、非手术和有效的基因编辑方法，需要了解新的基因编辑方法 针对慢性静脉曲张根本原因的治疗。在大约60%的CPVT患者中， RYR2通过心肌细胞内异常的钙离子转运导致心律失常。实验室已经表明，使用 CRISPR/Cas9敲除RYR2突变等位基因可有效预防室性心动过速 功能障碍的RyR2表达减少。这个应用程序的总体目标是改进治疗 CPVT患者。申请人产生的初步数据显示，直接针对一种疾病- 传统CRISPR/Cas9治疗引起CPVT小鼠模型突变可阻止起搏诱发的VT 但降低了RyR2的总表达。这一提议的中心假设是， CRISPR/Cas9基因编辑可通过特异性纠正致病突变位点、减少突变来治疗CPVT RyR2的表达，同时保持RyR2的总表达，正常化钙处理，并降低 对室上性心动过速易感性。这项研究的基本原理是，对有效性和特异性的理解 在纠正心脏突变方面的基因编辑可能会导致安全的新方法来治疗基因 心脏疾病。这一假设将通过以下具体目标进行检验：1)检验治疗潜力 在CPVT的IPSC-CM临床前模型中对人类RYR2突变的纠正和2)测试治疗 在慢性静脉曲张小鼠模型中纠正小鼠RYR2突变的可能性。为了确定目标1，我们交付 CRISPR/CAS9对IPSC的主要编辑向量，导出CM，并通过共焦和光测量功能 IPSC和3D心脏器官的薄层成像。为了确定目标2，我们将使用Lenti-CRISPR/Cas9素数 通过编辑载体治疗CPVT小鼠模型并测量长期心功能 超声心动图、心电图、程序性电刺激和分子分析。这项研究是 重要的是，它将促进RYR2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.