Uncovering Molecular Targets for Arrhythmogenic Cardiomyopathy Therapeutics

发现致心律失常性心肌病治疗的分子靶点

• 批准号: 10588199
• 负责人: Farah Sheikh
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10588199
• 关键词: Address; Adherens Junction; Adult; Age; Arrhythmogenic Right Ventricular Dysplasia; Biological Assay; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Complex; Connexins; Data; Defect; Desmosomes; Disease; Dose; Electrophysiology (science); Exhibits; Gene Mutation; Genes; Genetic; Genetic Diseases; Genome; Genomics; Heart; Heart Diseases; Human; In Vitro; Intercellular Junctions; Intervention; Knock-in Mouse; Life; Mechanics; Mediating; Methods; Modeling; Molecular; Molecular Target; Molecular Weight; Mus; Mutant Strains Mice; Mutate; Mutation; Neonatal; Pathogenesis; Pathogenicity; Patients; Physiological; Protein Analysis; Proteins; RNA; RNA Splicing; RNA Stability; RNA analysis; RNA-Binding Proteins; Splice-Site Mutation; Sudden Death; Technology; Testing; Therapeutic; Therapeutic Effect; Therapeutic Uses; Transcript; Viral; arrhythmogenic cardiomyopathy; base; base editing; base editor; design; disease-causing mutation; gene therapy; human disease; human model; in vivo; induced pluripotent stem cell derived cardiomyocytes; insight; man; mouse model; mutant; mutant mouse model; mutation correction; neonatal mice; novel; plakophilin 2; prevent; prime editing; prime editor; protein complex; protein degradation; restoration; scaffold; transcriptomics; young adult

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.

摘要 致心律失常性右心室发育不良/心肌病（ARVD/C）是一种无法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“ 在ARVD/C患者中40-50%的突变发现于桥粒（连接锚）基因中， 其中斑嗜蛋白-2（PKP 2）是最常突变的桥粒基因。有证据表明， RNA剪接可能是PKP 2患者遗传学驱动ARVD/C的关键机制。但没有 关于人类桥粒突变如何影响RNA剪接的机制， ARVD/C和什么形式的治疗方法在这些情况下会有影响。通过CRISPR-Cas9， 产生了一种新的小鼠模型，该模型在全球范围内含有影响RNA的人类PKP 2突变（IVS 10 -1 G>C）， 拼接PKP 2纯合子突变（PKP 2 Hom）小鼠选择性地显示ARVD/C的所有成年标志，包括 突然死亡RNA和测序分析揭示了低水平的较大PKP 2转录本，其保留了一个 内含子序列PKP 2 Hom心脏的蛋白质分析显示低水平的较高分子量PKP 2 突变体蛋白，其在内源性PKP 2不存在的情况下表达。增加野生型PKP 2的策略 在PKP 2突变的新生心肌细胞中，PKP 2和突变的PKP 2蛋白的剪接作用表明， 在早期ARVD/C中，单倍不足机制驱动细胞连接缺陷。PKP 2的靶向修复 新生PKP 2 Hom小鼠中的蛋白剂量在晚期ARVD/C中具有治疗潜力，因为它恢复了心脏 机械连接复合物和延长成年PKP 2 Hom小鼠的寿命。PKP 2 Hom小鼠提供了理想的测试 评估PKP 2恢复在经典患者中规避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， 作为纠正单碱基突变和解决ARVD/C“根本原因”的新方法， 有限的研究已经将该技术应用于疾病环境中的治疗用途。我们假设 PKP 2 RNA剪接突变足以通过影响剪接的机制驱动ARVD/C 对PKP 2蛋白剂量的影响。PKP 2靶向策略（基因治疗和引物碱基编辑指导） 校正）可用于治疗性改变ARVD/C。我们的目标是确定：（i）致病机制 PKP 2 RNA剪接突变驱动ARVD/C，（ii）早期和晚期PKP 2的影响和机制 在我们的新型PKP 2突变小鼠和人ARVD/C模型中的恢复，以及（iii）碱基编辑策略， 纠正PKP 2（IVS 10 -1 G>C）突变并评估其在我们新型PKP 2突变ARVD/C模型中的影响。