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）：少年神经元粘膜脂肪促脂肪促脂肪促脂肪（JNCL），也称为Batten病，是由CLN3基因突变引起的一种常染色体隐性疾病，全球范围内发生了12,500例活生生。它是一组溶酶体储存障碍的一部分 - 神经元的脂肪糖蛋白酶（NCLS） - 共同构成了最常见的儿童神经退行性疾病。 JNCL是一种毁灭性的疾病，导致视力丧失，运动和认知缺陷，癫痫发作，自身免疫性不规则和过早死亡。目前无法治愈JNCL，并且治疗仅限于减轻疾病的症状。目前，由于缺乏能够准确重现该疾病的多系统性质的动物模型，目前，改善了JNCL的疗法的发展受到限制。 JNCL的几种小鼠模型是通过靶向破坏鼠CLN3基因开发的，这些模型对于理解CLN3功能和JNCL疾病非常有价值。然而，没有一个小鼠模型完全复制人类疾病中观察到的复杂临床表现，更重要的是，没有人会发展出严重的神经系统型，这是人类JNCL疾病的标志。这可能是物种生理学，解剖学和发育差异的结果。相比之下，猪可以作为研究JNCL的更好模型，因为它们与人类相似。长期以来，猪已被用来对人类疾病进行建模，并被研究为人类异种移植的器官的来源。由于猪脑的发育和解剖学与人类的发育和解剖相比，与小鼠相比，猪CLN3基因的突变可能会导致许多在JNCL患者中观察到的许多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，该提案的最终目标是通过破坏CLN3基因来开发JNCL的猪模型。我们打算通过将基因靶向和体细胞核转移（SCNT）结合到两个步骤来完成这一目标。该建议概述了具有突变的CLN3等位基因的猪成纤维细胞的发展。将开发靶向载体的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患者中最常见的突变）。猪胎儿成纤维细胞将被带有CLN3靶向载体的病毒感染。我们生成正确靶向细胞的计划旨在最大化同源重组的频率，最大程度地减少随机整合并最大程度地减少收获靶细胞之前的细胞通道数量。随后的项目将使用这些细胞进行体细胞核转移，以产生以CLN3为目标的猪，然后进行表征和验证活性。 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功能障碍和JNCL的发病机理的后果，并开发和测试新的治疗和预防策略。