Development of a Porcine Model of Atherosclerosis

描述（由申请人提供）：动脉粥样硬化是心血管疾病的主要原因，心血管疾病是美国最常见的死亡原因。动脉粥样硬化的特征在于脂质、胆固醇、钙沉积物和细胞碎片在血管壁中的积聚，并导致斑块形成、动脉阻塞和流向器官的血流减少。这些斑块经常破裂，导致心肌梗塞、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。目前的治疗策略旨在通过药理学方法改变患者的生活方式/饮食和降低胆固醇。对于晚期病例，使用支架等医疗器械进行手术干预。虽然这些治疗方法使许多患有这种疾病的患者受益，但它们远非理想。其中一个原因是，没有一种药物或设备实际上是在一个模型系统中开发和测试的，可以准确地重现正在治疗的疾病。因此，早期临床前研究和人类药物试验之间存在显著差距。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疗法和干预措施的主要障碍。已经产生了几种小鼠模型，其基因突变对脂蛋白代谢很重要，虽然这些模型提供了信息，但它们未能发展出人类疾病典型的复杂动脉粥样硬化病变。与小鼠相反，猪心血管系统的生理学和解剖学与人类非常相似。事实上，猪长期以来一直被用作心血管疾病的模型，并且已经报道了在其LDL受体（LDLR）基因中具有天然发生的突变的猪，因此具有升高的LDL。虽然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，疾病的高变异性，其他研究人员的有限访问以及费用阻止其在研究界的广泛使用。因此，本项目的最终目标是开发和商业化基因靶向的猪动脉粥样硬化模型。我们在I期开发的LDLR胎儿成纤维细胞将用作体细胞核移植的核供体。将核移植胚胎转移至受体雌性进行妊娠。所得仔猪将具有一个靶向LDLR基因。我们将在分子和生化水平上对LDLR靶向猪进行表征。我们将确定LDL受体靶向猪的脂质和脂蛋白谱，并进行形态学分析，以确定动脉粥样硬化的存在和程度。最后，我们将建立繁殖群以产生LDLR-/-猪并扩大和繁殖殖民地。该项目将产生动脉粥样硬化的猪模型，这将为学术和行业研究人员提供更好地了解疾病并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昂贵而致命的疾病的新疗法。 公共卫生相关性：该提案具体概述了动脉粥样硬化基因工程猪模型的开发、表征和繁殖。该项目与NIH的使命相关，因为它将提供一种资源来刺激发现、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.