Development of a Porcine Model of Ataxia-Telangiectasia

描述(申请人提供)：共济失调-毛细血管扩张症(A-T)是一种多系统的隐性遗传性疾病，主要特征是早发性小脑性共济失调和毛细血管扩张，疾病名称由此而来。此外，患者还表现出许多其他临床症状，包括对癌症(淋巴瘤、白血病、脑瘤)的易感性增加，免疫缺陷，胰岛素抵抗糖尿病，染色体不稳定，对电离辐射敏感，易患支气管肺病，以及几乎完全没有胸腺。A-T是一种进行性并最终致命的疾病，大多数患者在20岁出头时死亡。目前对A-T的治疗主要针对症状的管理。物理和语言治疗可能会改善患者的日常生活，可以注射3-球蛋白来支持免疫系统。然而，目前还没有针对潜在缺陷的治疗方法。目前，由于缺乏完全和准确地概括这种疾病的多系统本质的动物模型，改进的A-T治疗方法的发展受到限制。通过靶向破坏小鼠ATM基因，已经建立了许多A-T的小鼠模型，并被证明对于研究ATM功能和A-T病的某些方面是非常有价值的。然而，没有一个单一的小鼠模型完全复制人类疾病中观察到的复杂临床症状，更重要的是，没有一个小鼠模型发展出严重的神经表型，这是人类A-T的标志。小鼠模型未能出现A-T的典型症状，可能是由于这两个物种之间的生理、解剖学和发育差异造成的。相比之下，猪可能是研究人类疾病的更好的模型，因为它们的发育、解剖学和生理学与人类的关系更密切。鉴于猪脑的发育和解剖更接近人类，而不是老鼠，猪ATM基因的突变可能会导致许多与A-T患者相同的神经变化。这项提议的最终目标是通过破坏ATM基因来开发和商业化A-T的猪模型。我们打算通过基因打靶和体细胞核移植(SCNT)相结合的两个步骤来实现这一点。这项建议特别概述了带有突变的ATM等位基因的猪成纤维细胞的发育。将构建基因靶向载体，在患者经常突变的区域扰乱内源性猪ATM基因。猪胎儿成纤维细胞将被携带ATM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。后续的工作将使用这些细胞进行体细胞核移植，以产生ATM靶标猪，并随后对猪进行鉴定和验证。这种动物模型将为学术和商业研究团体提供一个机会，以更好地了解ATM功能障碍的后果和A-T疾病的发病机制，并开发和测试新的治疗策略。 公共卫生相关性：项目简介本提案特别概述了带有突变的ATM等位基因的猪成纤维细胞的发展，作为迈向人类疾病新模型--共济失调-毛细血管扩张症的第一步。后续工作将使用这些细胞进行体细胞核移植，以产生受影响的猪，然后对动物模型进行表征和验证。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): Ataxia-Telangiectasia (A-T) is a multi-systemic, recessively inherited disorder characterized primarily by early onset cerebellar ataxia and telangiectasia, from which the disease name is derived. In addition, patients also exhibit a number of other clinical symptoms including increased susceptibility to cancer (lymphomas, leukemia, brain tumors), immunodeficiency, insulin-resistant diabetes, chromosomal instability, sensitivity to ionizing radiation, susceptibility to bronchopulmonary disease, and the nearly complete absence of a thymus. A-T is a progressive and ultimately fatal disease, with most patients dying in their early twenties. Current treatments for A-T are directed primarily toward the management of symptoms. Physical and speech therapy may improve the daily lives of patients, and 3-globulin injections can be given to support the immune system. However, no treatment is currently directed at the underlying defect. The development of improved therapies for A-T is currently limited by the lack of an animal model that fully and accurately recapitulates the multi-systemic nature of this disease. A number of mouse models of A-T have been developed by the targeted disruption of the mouse Atm gene and have proved invaluable for studying some aspects of ATM function and A-T disease. However, no single mouse model fully replicates the complex clinical symptoms observed in human disease, and more importantly, none of the mouse models develop the severe neurological phenotype that is the hallmark of human A-T. The failure of mouse models to develop the classical symptoms of A-T is likely the result of physiological, anatomical, and developmental differences between the two species. In contrast, pigs may serve as a better model in which to study human disease because their development, anatomy, and physiology are more closely related to that of humans. Given that the development and anatomy of the pig brain more closely resembles that of humans than mice, mutations in the porcine ATM gene may result in many of the same neurological changes that are observed in A-T patients. The ultimate goal of this proposal is to develop and commercialize a porcine model of A-T by disrupting the ATM gene. We intend to accomplish this in two steps by combining gene targeting and somatic cell nuclear transfer (SCNT). This proposal specifically outlines the development of porcine fibroblasts with mutated ATM alleles. Gene targeting vectors will be constructed to disrupt the endogenous porcine ATM gene in a region frequently mutated in patients. Porcine fetal fibroblasts will be infected with a virus carrying the ATM targeting vectors. 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 ATM-targeted pigs and the subsequent characterization and validation of the pigs. This animal model will provide the academic and commercial research communities an opportunity to better understand the consequences of ATM dysfunction and the pathogenesis of A-T disease, and to develop and test new therapeutic strategies. PUBLIC HEALTH RELEVANCE: Project Narrative This proposal specifically outlines the development of porcine fibroblasts with mutated ATM alleles as a first step towards a new model of the human disease, Ataxia-Telangiectasia. Subsequent work will use these cells for somatic cell nuclear transfer to produce affected pigs followed by characterization and validation of the animal model. 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.