A Patient-Specific hiPSC Model of Nilotinib-Induced Peripheral Artery Disease Pharmacogenomics

项目总结 外周动脉疾病(PAD)是动脉粥样硬化的一种亚型，在美国约有850万人受到影响 美国。PAD是全因死亡率的三倍，可与冠心病相媲美 增加死亡、心肌梗死和缺血性中风的风险。尼洛替尼是一种非常有效的治疗方法 用于慢性粒细胞白血病(CML)，但会导致与PAD一致的血压病理变化 服用一线和二线尼洛替尼的患者分别为26%和35%。即使在尼洛替尼治疗的患者中也会出现PAD 没有先前存在的心血管危险因素的患者，在某些情况下病情足够严重，需要 截肢。目前还没有工具来了解尼洛替尼诱导的PAD(N-PAD)或 先发制人地确定哪些患者可能对这种不良反应敏感，这意味着易感患者 只有在出现不可逆转的并发症后才能确定。人诱导多能干细胞 (HiPSCs)是研究药物不良反应个体间变异性的独特而有效的系统。 反应。HiPSC衍生物，包括心肌细胞、内皮细胞和血管平滑肌细胞， 以前被证明对药物诱导的和遗传的易感性都是患者特有的 表型。因为HiPSCs及其衍生物在基因上与患者完全相同 衍生，它们非常适合于N-PAD的药物基因组学研究，并可用于鉴定 并验证因果变异。这些变异将为临床基因筛查和机械性筛查提供信息 对N-PAD的理解。在本研究中，我们将建立N-PAD的体外模型。在目标1中，我们将在功能上 和生化特征的反应尼洛替尼来源的HiPSC来源的内皮细胞和血管 有无N-PAD患者的平滑肌细胞。我们预测这些细胞将重现患者- 对尼洛替尼治疗的特异性敏感性，并提供了一个模型，用来探索这种作用的机制。 在目标2中，我们将评估来自N-PAD患者的HiPSC来源的细胞对 尼洛替尼，以确定新的变种，然后将通过CRISPR/CAS9编辑进行验证。 这些目标的实现将建立PAD和动脉粥样硬化的体外模型 阐明N-PAD的机制并确定相关的变异以用于临床筛查。此外， 拟议的项目将为申请者的博士前培训提供平台，并允许开发 在实验设计和分析方面的专业知识，广泛的技术技能，以及 计算药物基因组学方法，同时也提高临床和专业技能。

PROJECT SUMMARY Peripheral artery disease (PAD), a subtype of atherosclerosis, affects approximately 8.5 million people in the United States. PAD confers a three-fold risk of all-cause mortality and is comparable to coronary heart disease in increased risk for mortality, myocardial infarction, and ischemic stroke. Nilotinib is a highly effective treatment for chronic myeloid leukemia (CML) but causes pathologic changes in blood pressure consistent with PAD in 26% and 35% of patients on first- and second-line nilotinib respectively. PAD occurs even in nilotinib-treated patients without pre-existing cardiovascular risk factors and in some cases is sufficiently severe to necessitate amputation. Currently no tools exist to understand the mechanism of nilotinib-induced PAD (N-PAD) or preemptively identify which patients may susceptible to this adverse effect, meaning that susceptible patients are identified only after they have developed irreversible complications. Human induced pluripotent stem cells (hiPSCs) constitute a unique and efficient system with which to study interindividual variability in adverse drug reactions. hiPSC derivatives, including cardiomyocytes, endothelial cells, and vascular smooth muscle cells, have previously been shown to recapitulate patient-specific susceptibility to both drug-induced and genetic phenotypes. Because hiPSCs and their derivatives are genetically identical to the patients from whom they are derived, they are well-suited to the study of the pharmacogenomics of N-PAD and can be used to both identify and validate causal variants. These variants will then inform clinical genetic screening as well as mechanistic understanding of N-PAD. In this study we will develop an in vitro model of N-PAD. In Aim 1 we will functionally and biochemically characterize response to nilotinib exposure in hiPSC-derived endothelial cells and vascular smooth muscle cells from patients with and without N-PAD. We predict that these cells will recapitulate patient- specific susceptibility to nilotinib treatment and provide a model with which to probe the mechanism of this effect. In Aim 2 we will assess the gene expression response of hiPSC-derived cells from patients with N-PAD to nilotinib in order to identify novel variants, which will then be validated through CRISPR/Cas9 editing. Accomplishment of these aims will establish an in vitro model for PAD and atherosclerosis in addition to elucidating the mechanism of N-PAD and identifying relevant variants for clinical screening. Additionally, the proposed project will provide a platform for the applicant's predoctoral training and allow for the development of expertise in experimental design and analysis, a broad repertoire of technical skills, and expertise in computational pharmacogenomics approaches while also enhancing clinical and professional skills.