Development of a Porcine Model of Ataxia-Telangiectasia

共济失调毛细血管扩张猪模型的建立

• 批准号: 8199181
• 负责人: Christopher Rogers
• 金额: $ 15.68万
• 依托单位: EXEMPLAR GENETICS, LLC
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8199181
• 关键词: ATM function; ATM gene; Affect; Alleles; Anatomy; Animal Model; Ataxia Telangiectasia; Brain; Brain Neoplasms; Breeding; Cell Count; Cells; Chromosomal Instability; Clinical; Communities; Complex; Defect; Development; Diabetes Mellitus; Disease; Early Onset Cerebellar Ataxia; Exhibits; Failure; Family suidae; Fibroblasts; Frequencies; Functional disorder; Future; Gene Targeting; Generations; Genes; Globulins; Goals; Harvest; Human; Immune system; Immunologic Deficiency Syndromes; Inborn Genetic Diseases; Individual; Injection of therapeutic agent; Insulin Resistance; Ionizing radiation; Lead; Malignant Neoplasms; Medical; Modeling; Mus; Mutate; Mutation; Names; Nature; Neurologic; Nuclear; Organ; Pathogenesis; Patients; Phase; Phenotype; Phosphotransferases; Physical therapy; Physiological; Physiology; Predisposition; Process; Proteins; Recombinant adeno-associated virus (rAAV); Research; Source; Speech Therapy; Symptoms; Testing; Thymus Gland; Validation; Virus; Work; Xenograft procedure; base; design; early onset; fetal; homologous recombination; human disease; improved; leukemia/lymphoma; loss of function; mouse model; novel therapeutics; nuclear transfer; somatic cell nuclear transfer; symptom management; vector

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.

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性疾病，其主要特征是早发性小脑性共济失调和毛细血管扩张，该疾病名称即由此而来。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、胰岛素抵抗性糖尿病、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 是一种进行性的、最终致命的疾病，大多数患者在二十出头时死亡。目前 A-T 的治疗主要针对症状的管理。物理和言语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，目前没有针对潜在缺陷的治疗方法。目前，由于缺乏能够充分、准确地概括该疾病的多系统性质的动物模型，A-T 改良疗法的开发受到限制。通过靶向破坏小鼠 Atm 基因开发了许多 A-T 小鼠模型，并已证明对于研究 ATM 功能和 A-T 疾病的某些方面具有不可估量的价值。然而，没有一种小鼠模型能够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，没有一种小鼠模型能够发展出人类 A-T 标志性的严重神经表型。小鼠模型未能出现 A-T 的典型症状可能是两个物种之间生理、解剖和发育差异的结果。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比小鼠更接近人类，猪 ATM 基因的突变可能会导致许多与 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 A-T 猪模型并将其商业化。我们打算通过结合基因靶向和体细胞核移植（SCNT）分两步来实现这一目标。该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育。将构建基因靶向载体来破坏患者体内频繁突变区域的内源性猪 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。我们生成正确目标细胞的计划旨在最大限度地提高同源重组的频率，最大限度地减少随机整合，并最大限度地减少收获目标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，以生产 ATM 靶向猪，并对猪进行后续表征和验证。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。 公共健康相关性：项目叙述该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育，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来生产受影响的猪，然后对动物模型进行表征和验证。该项目与 NIH 的使命相关，因为它将提供资源来刺激新诊断工具的发现、治疗应用和开发。