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(申请人提供)：幼年神经元蜡样脂褐素沉着症(JNCL)，也被称为巴顿病，是一种由CLN3基因突变引起的常染色体隐性遗传病，全球发病率为每12,500名活产儿中有1名。它是一组溶酶体储存障碍的一部分-神经元蜡样脂褐素增多症(NCLS)-共同构成了儿童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、运动和认知障碍、癫痫发作、自身免疫紊乱和过早死亡。目前还没有治愈JNCL的方法，治疗仅限于缓解疾病症状。目前，由于缺乏准确再现这种疾病的多系统本质的动物模型，JNCL的改进治疗方法的开发受到限制。通过靶向干扰小鼠的Cln3基因，已经建立了几种JNCL小鼠模型，这些模型对于了解CLN3的功能和JNCL病是有价值的。然而，没有一个小鼠模型完全复制了人类疾病中观察到的复杂临床表现，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JNCL疾病的标志。这很可能是由于物种在生理、解剖和发育方面的差异造成的。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。长期以来，猪一直被用来模拟人类疾病，并被研究为人类异种移植的器官来源。由于猪脑的发育和解剖更接近人类，而不是小鼠，猪CLN3基因的突变可能会导致许多与JNCL患者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，本方案的最终目的是通过破坏CLN3基因来建立猪的JNCL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。这项建议概述了带有突变的CLN3等位基因的猪成纤维细胞的发育。将开发基因靶向载体，从内源性CLN3基因的两个副本中删除外显子7和8(JNCL患者最常见的突变)。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。随后的一个项目将使用这些细胞进行体细胞核移植，以产生以CLN3为靶标的猪，然后进行鉴定和验证活动。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。