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Development of a Porcine Model of Duchenne Muscular Dystrophy

杜氏肌营养不良症猪模型的建立

基本信息

批准号：
8199195
负责人：
Christopher Rogers
金额：
$ 16.06万
依托单位：
EXEMPLAR GENETICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-18 至 2012-01-17
项目状态：
已结题

项目摘要

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.

描述(由申请人提供)： Duchenne肌营养不良症(DMD)是一种由DMD基因突变引起的X连锁隐性遗传病，发病率为3500例男婴中有1例。功能性肌营养不良蛋白的丧失会导致骨骼肌和心肌的进行性退化。尽管我们对这种疾病的了解有了很大的进步，在开发新的治疗方法方面也取得了进展，DMD仍然是一种致命的疾病。人们对DMD的大部分了解都来自于对缺乏dystrophin的动物的研究，特别是小鼠和狗的模型。虽然营养不良的小鼠对机制研究很有用，但它不能形成典型的DMD患者的肌肉无力表型。犬模型更能代表人类的DMD，但由于表型的极端变异性，很难进行研究。犬类模型还受到突变选择有限、巨额费用和社会接受问题的困扰。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、更一致地复制人类DMD临床表现的动物模型。我们的目标是在猪身上建立一种改进的DMD模型。我们认为，猪模型比现有模型有几个优势。基因打靶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出基因相同的营养不良猪，并可能减少表型严重程度的变异性。此外，与狗相比，猪的生产成本更低，更容易饲养，也不会像同伴动物那样面临同样的伦理问题。该项目的最终目标是开发和商业化以DMD为目标的猪，作为Duchenne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创建一个携带人类常见DMD突变的猪模型来实现这一点。这项建议特别概述了带有突变的DMD基因的猪成纤维细胞的发育。将构建基因靶向载体，以删除患者经常突变的区域中内源性猪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。后续工作将使用这些细胞进行体细胞核移植，以产生针对DMD的猪，随后进行表型鉴定和验证。这种动物模型将为学术和商业研究团体提供一个更好地了解DMD以及开发和测试新的治疗策略的机会公共卫生相关性：项目简介本提案特别概述了具有突变的DMD等位基因的猪成纤维细胞的发展，作为迈向Duchenne肌营养不良新模型的第一步。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，然后将其表征并验证为合适的模型。这个项目与美国国立卫生研究院的任务相关，因为它将提供一个资源来刺激发现、治疗应用和新诊断工具的开发。