Development of a Porcine Model of Juvenile Neuronal Ceroid Lipofuscinosis

幼年神经元蜡质脂褐质病猪模型的建立

• 批准号: 8455173
• 负责人: Christopher Rogers
• 金额: $ 22.49万
• 依托单位: EXEMPLAR GENETICS, LLC
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8455173
• 关键词: Adolescent; Alleles; Anatomy; Animal Model; Antibiotics; Autoimmune Process; Blindness; Brain; Breeding; CLN3 gene; Cell Count; Cells; Cessation of life; Childhood; Clinical; Cognitive deficits; Complex; Development; Disease; Exons; Failure; Family suidae; Fibroblasts; Frequencies; Functional disorder; Gene Targeting; Generations; Genes; Genomics; Goals; Harvest; Human; Incidence; Industry; Live Birth; Methods; Mission; Modeling; Mus; Mutate; Mutation; Nature; Neurodegenerative Disorders; Neurologic; Neuronal Ceroid-Lipofuscinosis; Nuclear; Organ; Pathogenesis; Patients; Phase; Phenotype; Physiological; Physiology; Production; Recombinant adeno-associated virus (rAAV); Research Personnel; Resources; Retinal Diseases; Seizures; Source; Southern Blotting; Spielmeyer-Vogt Disease; Symptoms; Testing; Validation; Virus; Vision; Visual system structure; Work; Xenograft procedure; arm; base; design; disease phenotype; fetal; homologous recombination; human disease; improved; motor deficit; mouse model; novel therapeutics; premature; public health relevance; recombinase; screening; somatic cell nuclear transfer; species difference; vector

DESCRIPTION (provided by applicant): Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is an autosomal recessive disorder caused by mutations in the CLN3 gene with a worldwide incidence of 1 in 12,500 live births. It is part of a group of lysosomal storage disorders - the neuronal ceroid lipofuscinoses (NCLs) - that together constitute the most common neurodegenerative diseases of childhood. JNCL is a devastating disease that results in vision loss, motor and cognitive deficits, seizures, autoimmune irregularities, and premature death. Currently there is no cure for JNCL, and treatments are limited to alleviating symptoms of disease. The development of improved therapies for JNCL is currently limited by the lack of an animal model that accurately recreates the multi-systemic nature of this disease. Several mouse models of JNCL have been developed via targeted disruption of the murine Cln3 gene, and these models have been valuable for understanding CLN3 function and JNCL disease. However, none of the mouse models fully replicate the complex clinical manifestations observed in the human disease, and more importantly, none develop the severe neurological phenotype that is the hallmark of human JNCL disease. This is likely the result of species differences in physiology, anatomy, and development. In contrast, pigs may serve as a better model in which to study JNCL because of their similarity to humans. Pigs have long been used to model human diseases and are being studied as a source of organs for human xenotransplantation. Because the development and anatomy of the porcine brain more closely resembles that of humans than mice, mutations in the porcine CLN3 gene may result in many of the same neurological changes that are observed in patients with JNCL. The similarities of the porcine visual system offer advantages for modeling the vision phenotype, as well. Therefore, the ultimate goal of this proposal is to develop a porcine model of JNCL by disrupting the CLN3 gene. We intend to accomplish this in two steps by combining gene-targeting and somatic cell nuclear transfer (SCNT). This proposal outlines the development of porcine fibroblasts with mutated CLN3 alleles. Gene targeting vectors will be developed to delete exons 7 and 8 (the most common mutation in JNCL patients) from both copies of the endogenous CLN3 gene. Porcine fetal fibroblasts will be infected with a virus carrying the CLN3 targeting vector. 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. A subsequent project will use these cells for somatic cell nuclear transfer to produce CLN3-targeted pigs followed by characterization and validation activities. The JNCL porcine model will provide academic and industry researchers an opportunity to better understand the consequences of CLN3 dysfunction and the pathogenesis of JNCL, and to develop and test new therapeutic and preventative strategies.

描述（由申请人提供）：青少年神经元蜡样质脂褐质沉着症（JNCL），也称为巴顿病，是一种由 CLN3 基因突变引起的常染色体隐性遗传疾病，全球发病率为 12,500 分之一的活产儿。它是一组溶酶体贮积症——神经元蜡样质脂褐素沉积症（NCL）——的一部分，它们共同构成了儿童期最常见的神经退行性疾病。 JNCL 是一种毁灭性的疾病，会导致视力丧失、运动和认知缺陷、癫痫发作、自身免疫异常和过早死亡。目前 JNCL 无法治愈，治疗仅限于缓解疾病症状。目前，由于缺乏能够准确再现这种疾病的多系统性质的动物模型，JNCL 改进疗法的开发受到限制。通过靶向破坏小鼠 Cln3 基因，开发了几种 JNCL 小鼠模型，这些模型对于了解 CLN3 功能和 JNCL 疾病非常有价值。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表现，更重要的是，没有一种小鼠模型能够发展出人类 JNCL 疾病标志性的严重神经表型。这可能是生理学、解剖学和发育方面物种差异的结果。相比之下，猪可能是研究 JNCL 的更好模型，因为它们与人类相似。猪长期以来一直被用来模拟人类疾病，并且正在研究作为人类异种移植的器官来源。由于猪脑的发育和解剖结构比小鼠更接近人类，因此猪 CLN3 基因的突变可能会导致许多与 JNCL 患者中观察到的相同的神经系统变化。猪视觉系统的相似性也为视觉表型建模提供了优势。因此，本提案的最终目标是通过破坏CLN3基因来开发JNCL的猪模型。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。该提案概述了具有突变 CLN3 等位基因的猪成纤维细胞的发育。将开发基因靶向载体，从内源 CLN3 基因的两个拷贝中删除外显子 7 和 8（JNCL 患者中最常见的突变）。猪胎儿成纤维细胞将被携带 CLN3 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。后续项目将使用这些细胞进行体细胞核移植来生产 CLN3 靶向猪，然后进行表征和验证活动。 JNCL 猪模型将为学术和行业研究人员提供一个机会，以更好地了解 CLN3 功能障碍的后果和 JNCL 的发病机制，并开发和测试新的治疗和预防策略。