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、隐性遗传疾病，主要以早发性小脑性共济失调和毛细血管扩张为特征，该疾病名称由此而来。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）的易感性增加、免疫缺陷、胰岛素抵抗型糖尿病、染色体不稳定、对电离辐射敏感、对支气管肺疾病的易感性以及胸腺几乎完全缺失。a - t是一种进行性且最终致命的疾病，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。物理和语言治疗可以改善患者的日常生活，并且可以注射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，由于缺乏能够完全准确地概括这种疾病的多系统性质的动物模型，改进A-T治疗方法的发展受到限制。通过靶向破坏小鼠Atm基因，已经建立了许多小鼠A- t模型，并证明了对研究Atm功能和A- t疾病的某些方面的宝贵价值。然而，没有一个单一的小鼠模型完全复制在人类疾病中观察到的复杂临床症状，更重要的是，没有一个小鼠模型表现出严重的神经表型，这是人类A-T的标志。小鼠模型未能出现A-T的经典症状，可能是两种物种之间生理、解剖和发育差异的结果。相比之下，猪可以作为研究人类疾病的更好模型，因为它们的发育、解剖和生理与人类更密切相关。考虑到猪大脑的发育和解剖结构比老鼠更接近人类，猪ATM基因的突变可能导致许多在A-T患者中观察到的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猪的a - t模型。我们打算通过结合基因靶向和体细胞核移植（SCNT）两步来实现这一目标。本提案特别概述了具有突变ATM等位基因的猪成纤维细胞的发展。构建基因靶向载体，破坏患者体内常发生突变的内源性猪ATM基因。携带ATM靶向载体的病毒会感染猪胎儿成纤维细胞。我们的目标细胞生成计划旨在最大化同源重组的频率，最小化随机整合，最小化目标细胞收获前的细胞传代次数。后续工作将使用这些细胞进行体细胞核移植，以生产atm靶向猪，并对猪进行表征和验证。该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。

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.