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。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有效疗法的主要障碍。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，但这种突变的温和性质、疾病的高度变异性以及其他研究人员获得的机会有限，阻碍了它在研究界的广泛使用。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。这项建议特别概述了LDLR基因被破坏的猪成纤维细胞的发育。构建基因打靶载体打乱LDLR的编码区。猪胎儿成纤维细胞将被携带LDLR靶向载体的病毒感染。我们的目标细胞生成计划旨在最大化同源重组的频率，最大限度地减少随机整合，并在目标细胞被收获之前最小化细胞传代的数量。随后的工作将使用这些细胞进行体细胞核移植，以产生动脉粥样硬化的猪模型。这种动物模型将为学术和工业研究团体提供一个机会，以更好地了解疾病及其发病机制，并开发和测试新的治疗和预防策略。 公共卫生相关性：这项提案特别概述了LDLR基因被破坏的猪成纤维细胞的发展，作为通向新的动脉粥样硬化模型的第一步。随后的工作将利用这些细胞进行体细胞核移植，以培育出胆固醇升高的猪。这种动物模型将为学术和工业研究团体提供一个机会，以更好地了解心血管疾病及其发病机制，并开发和测试新的治疗和预防策略。

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. 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 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 and the limited access by other researchers prevents 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. This proposal specifically outlines the development of porcine fibroblasts with a disrupted LDLR gene. A gene targeting vector will be constructed to disrupt the coding region of LDLR. Pig fetal fibroblasts will be infected with a virus carrying the LDLR targeting vector. Our plans for generating properly targeted cells are designed to maximize the frequency of homologous recombination, minimize random integration, and minimize the number of cell passages before targeted cells are harvested. Subsequent work will use these cells for somatic cell nuclear transfer to produce a porcine model of atherosclerosis. This animal model will provide academic and industrial research communities with an opportunity to better understand the disease and its pathogenesis and to develop and test new therapeutics and preventative strategies. PUBLIC HEALTH RELEVANCE: This proposal specifically outlines the development of porcine fibroblasts with a disrupted LDLR gene as a first step towards a new model of atherosclerosis. Subsequent work will use these cells for somatic cell nuclear transfer to produce pigs with elevated cholesterol. This animal model will provide academic and industrial research communities with an opportunity to better understand cardiovascular disease and its pathogenesis, and to develop and test new therapeutics and preventative strategies.