Development of a Porcine Model of Atherosclerosis

描述(申请人提供)：动脉粥样硬化是心血管疾病的主要原因，在美国是最常见的死亡原因。动脉粥样硬化的特征是脂质、胆固醇、钙沉积和细胞碎片在血管壁上积聚，并导致斑块形成、动脉阻塞和流向器官的血液减少。这些斑块经常破裂，导致心肌梗死、中风或死亡。主要危险因素包括血脂水平升高、高血压和糖尿病。目前的治疗策略是通过药物方法改变患者的生活方式/饮食并降低胆固醇。晚期病例使用支架等医疗器械进行外科干预。虽然这些治疗方法已经使许多患有这种疾病的患者受益，但它们远远不是理想的。一个原因是，没有任何药物或设备实际上是在模型系统中开发和测试的，这些模型系统可以准确地再现正在治疗的疾病。因此，早期临床前研究和人类药物试验之间存在着显著的差距。缺乏准确复制人类动脉粥样硬化所有表现的动物模型一直是开发这种致命疾病的有效治疗和干预措施的主要障碍。已经建立了几个具有脂蛋白新陈代谢重要基因突变的小鼠模型，虽然这些模型提供了信息，但它们未能形成人类疾病典型的复杂动脉粥样硬化病变。与老鼠相比，猪心血管系统的生理和解剖结构与人类非常相似。事实上，猪长期以来一直被用作心血管疾病的模型，有报道称，猪的低密度脂蛋白受体(LDLR)基因自然发生突变，因此具有高密度脂蛋白。尽管高胆固醇血症猪是一个有吸引力的模型，但突变的温和性质、疾病的高度变异性、其他研究人员获得的机会有限以及费用高昂，阻碍了它在研究界的广泛应用。因此，本项目的最终目标是开发一种基因靶向的猪动脉粥样硬化模型并将其商业化。我们在一期培养的LDLR胎儿成纤维细胞将被用作体细胞核移植的核供体。核移植胚胎将被移植给接受移植的雌性受孕女性。由此产生的仔猪将拥有一个靶向LDLR基因。我们将在分子和生化水平上对LDLR靶向的猪进行表征。我们将测定以低密度脂蛋白为靶标的猪的脂类和脂蛋白谱，并进行形态计量分析，以确定动脉粥样硬化的存在和程度。最后，我们将建立繁殖群来生产LDLR-/-猪，并扩大和繁殖群体。该项目将产生一个动脉粥样硬化的猪模型，为学术和行业研究人员提供一个更好地了解这种疾病以及开发和测试新的治疗和预防策略的机会。因此，这项工作将加速发现这种代价高昂且致命的疾病的新疗法。 公共卫生相关性：该提案特别概述了基因工程猪动脉粥样硬化模型的开发、表征和推广。这个项目与美国国立卫生研究院的任务相关，因为它将提供一个资源来刺激发现、治疗应用和新诊断工具的开发。

DESCRIPTION (provided by applicant): Atherosclerosis is the primary cause of cardiovascular disease, which is the most common cause of death in the United States. Atherosclerosis is characterized by the accumulation of lipids, cholesterol, calcium deposits, and cellular debris in vessel walls, and results in plaque formation, arterial obstruction, and diminished blood flow to organs. These plaques often rupture, causing myocardial infarction, stroke, or death. The main risk factors include elevated lipid levels, hypertension, and diabetes. Current treatment strategies are directed at changing patient lifestyle/diet and decreasing cholesterol via pharmacological methods. Surgical interventions with medical devices such as stents are used for advanced cases. While these therapeutic approaches have benefited many patients with this disease, they are far from ideal. One reason is that no drug or device is actually developed and tested in a model system that accurately recreates the disease being treated. Thus, there is a significant gap between early phase preclinical studies and human drug trials. The lack of an animal model that accurately replicates all of the manifestations of human atherosclerosis has been a major barrier to the development of effective therapies and interventions for this deadly disease. Several mouse models have been generated with mutations in genes important for lipoprotein metabolism, and while these models have been informative, they fail to develop the complex atherosclerotic lesions that are typical of the human disease. In contrast to mice, the physiology and anatomy of the porcine cardiovascular system closely resembles that of humans. In fact, pigs have long been used as models of cardiovascular disease, and pigs with naturally occurring mutations in their LDL receptor (LDLR) gene, and therefore possessing elevated LDL, have been reported. Although the hypercholesterolemic pig is an attractive model, the mild nature of the mutation, the high variability of the disease, the limited access by other researchers, and the expense prevent its wide use in the research community. Therefore, the ultimate goal of this project is to develop and commercialize a gene- targeted porcine model of atherosclerosis. LDLR fetal fibroblasts that we developed in Phase I will be used as nuclear donors for somatic cell nuclear transfer. Nuclear transfer embryos will be transferred to recipient females for gestation. Resulting piglets will have one targeted LDLR gene. We will characterize the LDLR- targeted pigs at the molecular and biochemical level. We will determine the lipid and lipoprotein profile in LDLR-targeted pigs and perform morphometric analysis to determine the presence and extent of atherosclerosis. Finally, we will establish breeding herds to generate LDLR-/- pigs and to expand and propagate the colony. This project will produce a porcine model of atherosclerosis that will provide academic and industry researchers with an opportunity to better understand the disease and to develop and test new therapeutics and preventative strategies. Thus, this work will accelerate the discovery of novel therapies for this costly and deadly disease. PUBLIC HEALTH RELEVANCE: This proposal specifically outlines the development, characterization and propagation of a genetically engineered porcine model of atherosclerosis. This project is relevant to the NIH's mission because it will provide a resource to stimulate discovery, therapeutic application, and the development of new diagnostic tools.