Uncovering Molecular Targets for Arrhythmogenic Cardiomyopathy Therapeutics

摘要 致心律失常性右室发育不良/心肌病(ARVD/C)是一种不可治愈的遗传性心脏病 导致年轻人和运动员猝死的疾病。ARVD/C被称为“一种疾病” 由于ARVD/C患者40%-50%的突变是在桥粒(连接锚)基因中发现的， 其中，蛋白亲和素2(PKP2)是最常突变的桥粒基因。有证据表明， RNA剪接可能是PKP2患者遗传学驱动ARVD/C的关键机制。 关于人类RNA剪接桥粒突变如何影响的模型和有限的机械性见解 ARVD/C以及什么形式的治疗在这些环境中会产生影响。通过CRISPR-CAS9 WE 建立了一种新的小鼠模型，该模型全局携带有影响RNA的人PKP2突变(IVS10-1G&gt；C) 拼接。PKP2纯合子突变(PKP2 Hom)小鼠选择性地表现出ARVD/C的所有成人特征，包括 猝死。RNA和测序分析显示，较大的PKP2转录本水平较低，保留了 内含子序列。PKP2人心脏的蛋白质分析显示，较高分子量的PKP2水平较低 在没有内源性PKP2的情况下表达的突变蛋白。增加野生型PKP2的策略 而在PKP2突变的新生心肌细胞中突变的PKP2蛋白表明剪接对PKP2的影响 单倍体功能不全导致ARVD/C早期细胞连接缺陷--PKP2靶向修复 新生PKP2Hom小鼠的蛋白质剂量在ARVD/C晚期具有治疗潜力，因为它恢复了心脏 机械连接复合体与成年PKP2Hom小鼠寿命延长。PKP2 Hom小鼠提供了理想的测试 评估PKP2修复对绕过经典患者ARVD/C的影响和机制的平台- 疾病早期和晚期的中心模型。主要的编辑(搜索和替换)策略已经出现 将老化作为纠正单碱基突变和解决ARVD/C的“根本原因”的新方法 有限的研究将这项技术应用于疾病环境中的治疗。我们假设 PKP2 RNA剪接突变足以通过影响剪接的机制驱动ARVD/C 对PKP2蛋白剂量的影响。PKP2靶向策略(基因治疗和基础编辑指导 可用于治疗改变ARVD/C。我们的目标是确定：(I)致病机制 PKP2 RNA剪接突变是如何驱动ARVD/C的，(Ii)早期和晚期PKP2的影响和机制 在我们新的PKP2突变小鼠和人类ARVD/C模型中的恢复，以及(Iii)碱基编辑策略 纠正PKP2(IVS10-1G&gt；C)突变，并评估其在我们新的PKP2突变ARVD/C模型中的影响。

Abstract Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an incurable genetic based cardiac disease that causes sudden death in young adults and athletes. ARVD/C is termed a “disease of the desmosome” as 40-50% of mutations in ARVD/C patients are found in desmosomal (junctional anchor) genes, with plakophilin-2 (PKP2) being the most frequently mutated desmosomal gene. Evidence suggests that altered RNA splicing may be a critical mechanism through which PKP2 patient genetics drive ARVD/C. However, no models and limited mechanistic insights exist into how human desmosomal mutations in RNA splicing impact ARVD/C and what form of therapeutics would be impactful in these settings. Through CRISPR-Cas9 we generated a novel mouse model globally harboring a human PKP2 mutation (IVS10-1 G>C) that impacts RNA splicing. PKP2 homozygous mutant (PKP2 Hom) mice selectively display all adult hallmarks of ARVD/C including sudden death. RNA and sequencing analyses revealed low levels of a larger PKP2 transcript that retains an intronic sequence. Protein analyses of PKP2 Hom hearts revealed low levels of a higher molecular weight PKP2 mutant protein that was expressed in the absence of endogenous PKP2. Strategies to increase wild type PKP2 and mutant PKP2 protein in PKP2 mutant neonatal cardiomyocytes suggested that splicing effects on PKP2 haploinsufficiency mechanistically drive cell junction deficits in early ARVD/C. Targeted restoration of PKP2 protein dose in neonatal PKP2 Hom mice had therapeutic potential in late ARVD/C as it restored cardiac mechanical junction complex and prolonged life in adult PKP2 Hom mice. PKP2 Hom mice provide an ideal test platform to assess the impact and mechanism of PKP2 restoration in circumventing ARVD/C in classic patient- centric models during early and late stages of disease. Prime editing (search-and-replace) strategies have come to age as novel methods to correct single base mutations and address the “root cause” of ARVD/C, though limited studies have applied this technology towards therapeutic use in disease settings. We hypothesize the PKP2 RNA splicing mutation is sufficient to drive ARVD/C through a mechanism impacting splicing consequences on PKP2 protein dose. PKP2 targeted strategies (gene therapy and prime base editor-directed correction) can be exploited to therapeutically alter ARVD/C. We aim to determine: (i) the pathogenic mechanism by which PKP2 RNA splicing mutations drive ARVD/C, (ii) the impact and mechanism of early and late PKP2 restoration in our novel PKP2 mutant mouse and human ARVD/C models, and (iii) a base editing strategy to correct the PKP2 (IVS10-1 G>C) mutation and assess its impact in our novel PKP2 mutant ARVD/C model.