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）：项目摘要/摘要Duchenne肌肉营养不良症（DMD）是由DMD基因突变引起的X连锁隐性疾病，其中3500个男性出生中有1个。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脏肌肉的进行性变性。尽管我们对这种疾病的理解以及新的治疗方法发展的进步取得了重大进展，但DMD仍然是一种致命的疾病。关于DMD的大部分知识来自研究肌营养不良的动物，尤其是鼠类和犬模型。虽然对机械研究有用，但营养不良小鼠无法发展患者DMD的肌肉无力表型。犬类模型更代表人类DMD，但由于极端的表型变异性而难以研究。犬种模型还遭受了有限的突变选择，大量费用和社会接受的问题。在这些模型中表现出希望的治疗策略未能将其转化为诊所。迫切需要一种更准确和始终如一地复制人类DMD临床表现的动物模型。我们的目标是在猪中创建改进的DMD模型。我们认为，猪模型比现有型号具有多个优势。猪现在可以提供基因靶向，并将为与患者相关的突变提供机会。猪克隆技术将允许产生遗传上相同的营养不良猪，并可能导致表型严重程度的变异性降低。此外，猪的生产价格便宜，而不是狗更容易居住，并且不会面临与伴侣动物相同的道德问题。该项目的最终目标是开发和商业化靶向DMD的猪作为Duchenne肌肉营养不良的模型。我们打算通过结合基因靶向和体细胞核转移来实现这一目标，从而创建具有常见人DMD突变的猪模型。该建议专门概述了具有突变的DMD基因的猪成纤维细胞的发展。将构建靶向载体的基因，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带有DMD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。随后的工作将使用这些细胞进行体细胞核转移，以产生针对DMD的猪，然后进行表型表征和验证。该动物模型将为学术和商业研究社区提供更好地了解DMD并制定和测试新的治疗策略的机会公共卫生相关性：该建议的项目叙述专门概述了具有突变的DMD等位基因的猪成纤维细胞的发展，这是朝着新的Duchenne肌肉营养不良模型迈出的第一步。随后的工作将使用这些细胞进行体细胞核转移，以产生营养不良的猪，然后将其表征和验证为适当的模型。该项目与NIH的使命相关，因为它将提供刺激发现，治疗应用和开发新诊断工具的资源。