Novel Therapeutic Strategies in the Understanding of Systemic Amyloid Disease

项目摘要 家族性甲状腺素运载蛋白淀粉样变性（ATTR）是一种破坏性的多系统蛋白质折叠障碍， 甲状腺素运载蛋白（TTR）基因中超过100种可能的突变。在这种疾病中，TTR错误折叠， 并以浓度依赖性方式在下游靶器官（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），从诊断到死亡的平均时间仅为5-10年。 ATTR患者的当前护理标准，包括肝移植和小分子TTR 稳定剂是非常有限的;并非所有患者都是手术的候选人，存在大量供体器官缺陷， 许多患者对动力学稳定剂是难治的。更好地了解疾病的病因以及替代疗法 治疗选择对于对抗系统性淀粉样蛋白紊乱是必要的。 问题是，该疾病的多组织性质使得难以在体外进行研究，而目前还没有 动物模型准确地再现了ATTR病理学。为了克服这些局限性，我们的实验室 开发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iPSC分化成产生突变TTR的效应细胞（肝细胞样细胞）。调节 然后在这些细胞上制备培养基以（1）分析分泌的TTR种类的类型和数量和（2）分析剂量 靶细胞（iPSC衍生的心肌细胞和神经元）以测定所得毒性和所提出的化合物的功效。 治疗学 使用我们的遗传易处理的模型，我们希望改善目前的ATTR治疗模式。 重要的是，研究表明，通过肝移植或肝移植降低不稳定TTR的血清水平， 应激反应蛋白质折叠机制的激活降低了靶器官毒性。有了这种洞察力 和我们的基于iPSC的ATTR模型，我们将检验以下假设： 内源性蛋白质折叠机制的激活将证明对ATTR有治疗作用。我们建议评估这一点 通过两个目标的假设。首先，为了克服位点特异性基因编辑方法的局限性， 治疗ATTR，我们将开发一种通用的基因校正策略，改善所有TTR遗传性病变。在 第二，我们将利用基因组学方法激活未折叠蛋白反应（UPR）中的ATF 6通路， 本发明涉及选择性减少不稳定的毒性TTR的产生的药理学方法。在这两种方法中， TTR种类的分泌及其对患者匹配的iPSC衍生的靶细胞（神经元和 心肌细胞）毒性。 本文所述的我们的基于iPSC的模型允许蛋白质生物化学的前所未有的耦合， 和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允许 更好地理解和治疗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.