Intronic CRISPR Repair in a Preclinical Model of Noonan Syndrome-Associated Cardiomyopathy
Intronic CRISPR Repair in a Preclinical Model of Noonan Syndrome-Associated Cardiomyopathy
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DOI：
10.1161/circulationaha.119.044794
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发表时间：
2020-09-15
期刊：
影响因子：
37.8
通讯作者：
Cyganek, Lukas
中科院分区：
文献类型：
--
作者：
Hanses, Ulrich;Kleinsorge, Mandy;Cyganek, Lukas
背景：Noonan综合征(NS)是一种多系统发育障碍，以常见的、临床可变的症状为特征，如典型的面部畸形、身材矮小、发育迟缓、智能障碍以及心肌肥大。其潜在的机制是Ras丝裂原激活的蛋白激酶信号通路的功能获得。然而，我们对心肌病的病理生理改变和机制，特别是相关的心肌病的了解仍然有限，缺乏有效的治疗选择。方法：在这里，我们介绍了一个有两个兄弟姐妹的家庭，表现为常染色体隐性形式的NS伴大量肥厚性心肌病，临床上最常见的症状是亮氨酸拉链样转录调节因子1(LZTR1)的双等位基因突变引起的。我们从受影响的同胞中培养出了诱导多能干细胞来源的心肌细胞，并从分子和功能水平上研究了患者特有的心肌细胞。结果：患者诱导的多能干细胞来源的心肌细胞重现了肥大表型，并发现了LZTR1功能障碍、RAS丝裂原激活蛋白激酶信号过度活性、肥大基因反应和细胞肥大之间迄今尚未描述的因果联系。钙通道阻断和MEK抑制可以预防某些疾病特征，为NS患者临床使用这些药物提供了分子基础，但可能不是一个可持续的治疗选择。在概念验证的方法中，我们探索了临床上可翻译的CRISPR(簇状规则间隔短回文重复)修复，并展示了对肥大表型的挽救。结论：我们的研究揭示了来自携带LZTR1双等位基因变异的NS患者的患者特异性诱导多能干细胞来源的心肌细胞的人类心脏发病机制，并鉴定了一种独特的疾病特异性蛋白质组特征。此外，我们认为内含子CRISPR修复是治疗NS相关肥厚型心肌病的一种个体化和临床可翻译的治疗策略。
Background: Noonan syndrome (NS) is a multisystemic developmental disorder characterized by common, clinically variable symptoms, such as typical facial dysmorphisms, short stature, developmental delay, intellectual disability as well as cardiac hypertrophy. The underlying mechanism is a gain-of-function of the RAS-mitogen-activated protein kinase signaling pathway. However, our understanding of the pathophysiological alterations and mechanisms, especially of the associated cardiomyopathy, remains limited and effective therapeutic options are lacking. Methods: Here, we present a family with two siblings displaying an autosomal recessive form of NS with massive hypertrophic cardiomyopathy as clinically the most prevalent symptom caused by biallelic mutations within the leucine zipper-like transcription regulator 1 (LZTR1). We generated induced pluripotent stem cell-derived cardiomyocytes of the affected siblings and investigated the patient-specific cardiomyocytes on the molecular and functional level. Results: Patients' induced pluripotent stem cell-derived cardiomyocytes recapitulated the hypertrophic phenotype and uncovered a so-far-not-described causal link between LZTR1 dysfunction, RAS-mitogen-activated protein kinase signaling hyperactivity, hypertrophic gene response and cellular hypertrophy. Calcium channel blockade and MEK inhibition could prevent some of the disease characteristics, providing a molecular underpinning for the clinical use of these drugs in patients with NS, but might not be a sustainable therapeutic option. In a proof-of-concept approach, we explored a clinically translatable intronic CRISPR (clustered regularly interspaced short palindromic repeats) repair and demonstrated a rescue of the hypertrophic phenotype. Conclusions: Our study revealed the human cardiac pathogenesis in patient-specific induced pluripotent stem cell-derived cardiomyocytes from NS patients carrying biallelic variants inLZTR1and identified a unique disease-specific proteome signature. In addition, we identified the intronic CRISPR repair as a personalized and in our view clinically translatable therapeutic strategy to treat NS-associated hypertrophic cardiomyopathy.