Development of a Porcine Model of Duchenne Muscular Dystrophy

描述（由申请人提供）： 摘要杜氏肌营养不良症（Duchenne muscular dystrophy，DMD）是由DMD基因突变引起的X连锁隐性遗传疾病，发病率为1/3500。随之而来的功能性肌营养不良蛋白的丧失导致骨骼肌和心肌的进行性变性。尽管我们对这种疾病的理解和新治疗方法的开发取得了重大进展，但DMD仍然是一种致命的疾病。关于DMD的大部分知识来自于对肌营养不良蛋白缺陷动物的研究，特别是小鼠和犬模型。虽然可用于机制研究，但营养不良小鼠不能发展出DMD患者典型的肌无力表型。犬模型更能代表人DMD，但由于极端的表型变异性而难以研究。犬模型也遭受有限的突变选择，显着的费用，和社会接受的问题。在这些模型中显示出希望的治疗策略未能转化为临床。迫切需要一种更准确和一致地复制人类DMD临床表现的动物模型。我们的目标是在猪中创建DMD的改进模型。我们相信猪模型比现有模型具有几个优势。基因靶向现在可以在猪身上使用，这将为设计与患者相关的突变提供机会。猪克隆技术将允许生产遗传上相同的营养不良猪，并可以减少表型严重程度的变异性。此外，猪的生产成本较低，比狗更容易饲养，也不像伴侣动物那样面临同样的道德问题。该项目的最终目标是开发和商业化DMD靶向猪作为杜氏肌营养不良症的模型。我们打算通过结合基因打靶和体细胞核移植来实现这一目标，以创建一个携带常见人类DMD突变的猪模型。该提案具体概述了具有突变DMD基因的猪成纤维细胞的开发。将构建基因靶向载体以缺失患者中频繁突变的区域中的内源性猪DMD基因的一部分。猪胎儿成纤维细胞将被携带DMD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。后续工作将使用这些细胞进行体细胞核移植，以产生DMD靶向猪，然后进行表型表征和验证。这种动物模型将为学术界和商业研究界提供一个更好地了解DMD并开发和测试新的治疗策略的机会 公共卫生相关性： 项目叙述本提案具体概述了具有突变DMD等位基因的猪成纤维细胞的开发，作为杜氏肌营养不良症新模型的第一步。随后的工作将使用这些细胞进行体细胞核移植，以产生营养不良的猪，然后将其作为适当的模型进行表征和验证。该项目与NIH的使命相关，因为它将提供一种资源来刺激发现、治疗应用和新诊断工具的开发。

DESCRIPTION (provided by applicant): Project Summary/Abstract Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene with a prevalence of 1 in 3500 male births. The consequent loss of functional dystrophin results in the progressive degeneration of skeletal and cardiac muscle. Despite significant progress in our understanding of this disease and advances in the development of new therapeutic approaches, DMD remains a fatal disease. Much of what is known about DMD has come from studying dystrophin-deficient animals, particularly murine and canine models. While useful for mechanistic studies, dystrophic mice fail to develop the muscle weakness phenotype that is typical of DMD in patients. The canine models are more representative of human DMD, but are difficult to study due to extreme phenotypic variability. The canine models also suffer from a limited choice of mutations, significant expense, and social acceptance concerns. Therapeutic strategies that have shown promise in these models have failed to be translated to the clinic. An animal model that more accurately and consistently replicates the clinical manifestations of human DMD is sorely needed. Our objective is to create an improved model of DMD in the pig. We believe a porcine model offers several advantages over the existing models. Gene targeting is now available in pigs and would provide an opportunity to engineer patient-relevant mutations. Porcine cloning technology would allow the production of genetically identical dystrophic pigs and could yield reduced variability in phenotype severity. Also, pigs are less expensive to produce and easier to house than dogs and don't face the same ethical concerns as companion animals. The ultimate goal of this project is to develop and commercialize DMD-targeted pigs as a model of Duchenne muscular dystrophy. We intend to accomplish this by combining gene targeting and somatic cell nuclear transfer to create a porcine model harboring a common human DMD mutation. This proposal specifically outlines the development of porcine fibroblasts with a mutated DMD gene. Gene targeting vectors will be constructed to delete a portion of the endogenous porcine DMD gene in a region frequently mutated in patients. Porcine fetal fibroblasts will be infected with a virus carrying the DMD 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 DMD-targeted pigs, followed by phenotypic characterization and validation. This animal model will provide the academic and commercial research communities an opportunity to better understand DMD and to develop and test new therapeutic strategies PUBLIC HEALTH RELEVANCE: Project Narrative This proposal specifically outlines the development of porcine fibroblasts with mutated DMD alleles as a first step towards a new model of Duchenne muscular dystrophy. Subsequent work will use these cells for somatic cell nuclear transfer to produce dystrophic pigs that will then be characterized and validated as an appropriate 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.