Development of a Porcine Model of Ataxia-Telangiectasia

描述（由申请人提供）：共济失调-毛细血管扩张症（A-T）是一种多系统、复发性遗传性疾病，主要特征为早发性小脑共济失调和毛细血管扩张症，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、脑肿瘤）、免疫缺陷、胰岛素抵抗性糖尿病、染色体不稳定性、电离辐射敏感性、支气管肺结核病易感性和几乎完全缺乏胸腺的易感性增加。A-T是一种进行性和最终致命的疾病，大多数患者在20岁出头时死亡。目前A-T的治疗主要针对症状的管理。物理和语言治疗可以改善患者的日常生活，3-球蛋白注射可以支持免疫系统。然而，目前没有针对潜在缺陷的治疗。A-T的改进疗法的开发目前受到缺乏完全和准确地概括这种疾病的多系统性质的动物模型的限制。通过靶向破坏小鼠Atm基因，已经开发了许多A-T小鼠模型，并且已经证明对于研究ATM功能和A-T疾病的某些方面是非常宝贵的。然而，没有一个小鼠模型完全复制了在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系统表型，这是人类A-T的标志。小鼠模型未能出现A-T的经典症状可能是两个物种之间生理学、解剖学和发育差异的结果。相比之下，猪可能是研究人类疾病的更好模型，因为它们的发育，解剖学和生理学与人类更密切相关。鉴于猪脑的发育和解剖结构与人类比小鼠更相似，猪ATM基因的突变可能导致许多与A-T患者相同的神经系统变化。该提案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。我们打算通过结合基因打靶和体细胞核移植（SCNT）两步完成这一目标。该提案具体概述了具有突变ATM等位基因的猪成纤维细胞的开发。将构建基因靶向载体以破坏患者中频繁突变的区域中的内源性猪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。我们用于产生适当靶向细胞的计划旨在最大化同源重组的频率，最小化随机整合，并最小化收获靶向细胞之前的细胞传代次数。后续工作将使用这些细胞进行体细胞核移植，以产生ATM靶向猪，并对猪进行后续表征和验证。这种动物模型将为学术和商业研究界提供一个机会，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：项目叙述本提案具体概述了具有突变ATM等位基因的猪成纤维细胞的开发，作为人类疾病共济失调-毛细血管扩张症新模型的第一步。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。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): 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.