Novel Therapeutic Strategies in the Understanding of Systemic Amyloid Disease

项目总结 家族性甲状腺激素转运蛋白淀粉样变性是一种破坏性的多系统蛋白质折叠障碍，其结果是 来自100多种可能的转甲状腺激素(TTR)基因突变。在这种疾病中，TTR错误折叠，是从 肝脏，并以浓度依赖的方式在下游靶器官如 如心脏和/或外周神经系统。ATTR在疾病中表现出极端的突变依赖性变异 表型(如受影响的靶器官和严重程度)，确诊至死亡的平均时间仅为5-10年。 目前对ATTR患者的护理标准，包括肝移植和小分子TTR 稳定剂，是高度有限的；并不是所有的患者都适合手术，存在大量的供体器官缺陷，以及 许多患者对动力稳定剂无效。更好地了解疾病病因和替代方案 治疗方案对于对抗系统性淀粉样蛋白紊乱是必要的。 问题是，这种疾病的多组织性质使得在体外进行研究变得困难，而目前还没有 动物模型准确地概括了Attr的病理学。为了克服这些限制，我们的实验室已经 发展了一种新的基于诱导多能干细胞(IPSC)的模型来研究Attr。在我们的平台上，病人- 衍生的IPSCs被分化成效应细胞(肝细胞样细胞)，产生突变的TTR。条件性的 然后在这些细胞上制备培养基以(1)分析TTR物种分泌的类型和数量，(2)剂量 靶细胞(IPSC来源的心肌细胞和神经元)，以分析由此产生的毒性和建议的疗效 治疗学。 使用我们的遗传易处理模型，我们希望改进目前对ATTR的治疗范例。 重要的是，研究表明，通过肝移植或降低血清中不稳定的TTR水平 应激反应蛋白折叠机械的激活降低了靶器官的毒性。用这种洞察力武装起来 和我们基于IPSC的ATTR模型，我们将检验这样一个假设，即TTR异常表达的中断或 内源性蛋白质折叠机制的激活将被证明是对ATTR的治疗。我们建议对此进行评估 通过两个目的提出假设。首先，为了克服针对特定部位的基因编辑方法的局限性 在治疗ATTR时，我们将开发一种通用的基因纠正策略，改善所有TTR基因损伤。在……里面 第二，我们将利用基因组和蛋白质激活未折叠蛋白反应(UPR)的ATF6途径 选择性地减少不稳定的、有毒的TTR的产生的药理学方法。在这两种方法中， TTR族的分泌及其对患者相合的IPSC来源的靶细胞(神经元和 心肌细胞)毒性将被评估。 我们基于IPSC的模型，在这里描述，允许前所未有的蛋白质生物化学的耦合 以及基于基因组的方法来研究系统性淀粉样变性的新方面。在这里获得的洞察力将使 以更好地了解和治疗ATTR和其他蛋白质折叠障碍。

PROJECT SUMMARY Familial transthyretin amyloidosis (ATTR) is a devastating multi-systemic protein folding disorder that results from over 100 possible mutations in the transthyretin (TTR) gene. In the disease, TTR misfolds, is secreted from the liver, and aggregates extracellularly in a concentration-dependent manner at downstream target organs such as the heart and/or peripheral nervous system. ATTR exhibits extreme mutation-dependent variation in disease phenotype (e.g. target organ affected and severity) with an average time of diagnosis to death of only 5-10 years. The current standards of care for patients with ATTR, including liver transplantation and small molecule TTR stabilizers, are highly limited; not all patients are candidates for surgery, large donor organ deficits exist, and many patients are refractory to kinetic stabilizers. A better understanding of disease etiology as well as alternative treatment options are necessary to combat systemic amyloid disorders. Problematically, the multi-tissue nature of the disease makes it difficult to study in vitro, while no current animal model accurately recapitulates ATTR pathology. To combat these limitations, our laboratory has developed a novel, induced pluripotent stem cell (iPSC)-based model for studying ATTR. In our platform, patient- derived iPSCs are differentiated into effector cells (hepatocyte-like cells) that produce mutant TTR. Conditioned media is then prepared on these cells to (1) analyze the type and quantity of TTR species secreted and (2) dose target cells (iPSC-derived cardiomyocytes and neurons) to assay resulting toxicity and the efficacy of proposed therapeutics. Using our genetically tractable model, we look to improve the current therapeutic paradigm for ATTR. Importantly, studies show that reducing serum levels of destabilized TTR through liver transplantation or activation of stress-responsive protein folding machinery reduces target organ toxicity. Armed with this insight and our iPSC-based ATTR model, we will test the hypothesis that disruption of aberrant TTR expression or activation of endogenous protein folding machinery will prove therapeutic for ATTR. We propose to evaluate this hypothesis through two Aims. In the first, to overcome limitations of site-specific gene editing approaches for treating ATTR, we will develop a universal gene correction strategy ameliorative of all TTR genetic lesions. In the second, we will activate the ATF6 pathway of the unfolded protein response (UPR) using genomic and pharmacological approaches to selectively decrease production of destabilized, toxic TTR. In both methods, secretion of TTR species and their impact on patient-matched iPSC-derived target cell (neuron and cardiomyocyte) toxicity will be evaluated. Our iPSC-based model, described herein, allows for the unprecedented coupling of protein biochemistry and genomic-based approaches to study novel aspects of systemic amyloidoses. Insight gained here will allow for better understanding of and therapeutics for ATTR and other protein folding disorders.