Development of a Porcine Model of Juvenile Neuronal Ceroid Lipofuscinosis

描述（由申请方提供）：青少年神经元蜡样质脂褐质沉积症（JNCL），也称为Batten病，是一种由CLN 3基因突变引起的常染色体隐性遗传疾病，全球发病率为1/12，500活产婴儿。它是一组溶酶体贮积症的一部分-神经元蜡样脂褐质沉积症（NCL）-共同构成了儿童最常见的神经退行性疾病。JNCL是一种破坏性疾病，可导致视力丧失、运动和认知缺陷、癫痫发作、自身免疫性异常和过早死亡。目前，JNCL没有治愈方法，治疗仅限于缓解疾病症状。JNCL的改进疗法的开发目前受到缺乏准确再现这种疾病的多系统性质的动物模型的限制。通过靶向破坏小鼠Cln 3基因，已经开发了几种JNCL小鼠模型，这些模型对于理解CLN 3功能和JNCL疾病很有价值。然而，没有一个小鼠模型完全复制了在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系统表型，这是人类JNCL疾病的标志。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。长期以来，猪一直被用来模拟人类疾病，并被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与人类比小鼠更相似，因此猪CLN 3基因的突变可能导致许多与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，本提案的最终目标是通过破坏CLN 3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）分两步实现这一目标。该提议概述了具有突变的CLN 3等位基因的猪成纤维细胞的发展。将开发基因靶向载体，以从内源性CLN 3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CLN 3靶向载体的病毒感染。我们用于产生适当靶向细胞的计划旨在最大化同源重组的频率，最小化随机整合，并最小化收获靶向细胞之前的细胞传代次数。随后的项目将使用这些细胞进行体细胞核移植，以生产CLN 3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CLN 3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。

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.