Development of a Humanized Pig Model of Cystic Fibrosis

描述（由申请人提供）：囊性纤维化（CF）是由囊性纤维化跨膜传导调节因子（CFTR）基因突变引起的。CFTR基因编码一个氯离子通道，调节离子流过上皮细胞膜。大多数CF患者有一种突变，删除苯丙氨酸508 (？F508)，并阻止CFTR被正确处理并传递到细胞膜。CFTR未能到达肺细胞表面会破坏气道表面液体的组成和数量，并通过尚未完全了解的机制，引发一系列事件，导致慢性气道感染、炎症和肺破坏。尽管CF患者的生活得到了改善，但它仍然是一种致命的疾病，目前的治疗主要针对继发性表现，而不是潜在的缺陷。几项研究表明？F508-CFTR通道在实验输送到细胞膜时仍保持氯离子通道功能。因此，？F508-CFTR通道已成为药物干预的重要治疗靶点。然而，有两个障碍限制了新疗法的进展。首先，缺乏发展为肺部疾病的动物模型是了解疾病发病机制和开发有效治疗方法的主要障碍。敲除CFTR的小鼠和？F508突变已被发现，但它们没有表现出导致大多数患者发病率和死亡率的气道感染和炎症。第二，？其他物种的F508-CFTR蛋白与人类的F508-CFTR蛋白在加工过程中存在差异。因此，研究CF和开发新的治疗方法需要一种新的表达人类F508-CFTR的动物模型。在本应用中，我们建议生成这样一个模型。我们将在这个项目中使用猪，因为与小鼠相比，猪的肺和气道的结构、生理和生物化学与人类非常相似。该项目的最终目标是开发并商业化一种携带人类CFTR基因的CF猪模型。F508突变。“人性化”？F508猪将是开发和测试CF药物的理想模型，也是回答CF发病机制的长期问题的有价值的工具。该提案概述了这样一个猪的发展，首先稳定地引入整个人类CFTR基因承载？F508突变进入缺乏内源性猪CFTR表达的猪细胞基因组。这些转基因细胞将被用作体细胞核移植的核供体。由此产生的猪将进行分子、生物化学和电生理学表征，以确定？F508-CFTR的表达及功能。为了验证人类？F508-CFTR转基因猪将作为测试新疗法的相关模型，猪气道培养将被建立，已知的化合物将部分挽救？将对人体组织中的F508-CFTR进行检测。该项目将产生一个动物模型，为CF工业界和学术界提供一个机会，以更好地了解该疾病及其发病机制，并开发和测试新的治疗方法和预防策略。因此，这项工作将加速这种致命疾病的新疗法的发现。公共卫生相关性：该项目将产生一个动物模型，为囊性纤维化学术界和工业界提供一个机会，以更好地了解这种疾病及其发病机制，并开发和测试新的治疗方法和预防策略。因此，这项工作将加速这种致命疾病的新疗法的发现。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene encodes a chloride channel that regulates ion flow across epithelial cell membranes. Most CF patients have a mutation that deletes phenylalanine 508 (?F508) and prevents CFTR from being properly processed and delivered to cell membranes. Failure of CFTR to reach the cell surface in the lung disrupts the composition and quantity of the airway surface liquid, and through mechanisms not yet fully understood, begins a cascade of events that leads to chronic airway infection and inflammation and lung destruction. Despite improvements in the lives of patients with CF, it remains a lethal disease, and current treatments are directed at secondary manifestations rather than the underlying defect. Several studies have demonstrated that the ?F508-CFTR channel retains chloride channel function when it is experimentally delivered to the cell membrane. Thus, the ?F508-CFTR channel has become an important therapeutic target for pharmacological intervention. However, two barriers limit progress toward new treatments. First, lack of an animal model that develops lung disease is a major impediment to understanding disease pathogenesis and the development of effective therapies. Mice with CFTR knockouts and with the ?F508 mutation have been developed, but they fail to exhibit the airway infection and inflammation that cause most of the morbidity and mortality in patients. Second, the ?F508-CFTR protein from other species shows differences in its processing compared to human ?F508-CFTR. Thus, studies focused on understanding CF and developing new treatments require a new animal model expressing the human ?F508-CFTR. In this application, we propose to produce such a model. We will use pigs for this project, because in contrast to mice, the structure, physiology, and biochemistry of their lungs and airways closely resemble those of humans. The ultimate goal of this project is to develop and commercialize a porcine model of CF carrying a human CFTR transgene bearing the common ?F508 mutation. A "humanized" ?F508 pig would be the ideal model in which to develop and test CF drugs and a valuable tool for answering persistent questions about CF pathogenesis. This proposal outlines the development of such a pig by first stably introducing the entire human CFTR gene bearing the ?F508 mutation into the genome of pig cells lacking endogenous pig CFTR expression. The transgenic cells will then be used as nuclear donors for somatic cell nuclear transfer. The resulting pigs will be characterized molecularly, biochemically, and electrophysiologically to determine ?F508-CFTR expression and function. To validate the human ?F508-CFTR transgenic pig as a relevant model in which to test new therapies, pig airway cultures will be established, and compounds known to partially rescue the processing of ?F508-CFTR in human tissues will be tested. This project will generate an animal model that will provide the CF industrial and academic community with an opportunity to better understand the disease and its pathogenesis and to develop and test new therapeutics and preventative strategies. Thus, this work will accelerate the discovery of novel therapies for this lethal disease. PUBLIC HEALTH RELEVANCE: This project will generate an animal model that will provide the cystic fibrosis academic and industrial community with an opportunity to better understand the disease and its pathogenesis and to develop and test new therapeutics and preventative strategies. Thus, this work will accelerate the discovery of novel therapies for this lethal disease.