Engineering a CFTR Deficient Porcine Model of CF

设计 CFTR 缺陷猪 CF 模型

• 批准号: 7345636
• 负责人: SCOTT M. O'GRADY
• 金额: $ 24.11万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2009-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7345636
• 关键词: Address; Adult; Animal Model; Animals; Biological Models; Blood Vessels; Characteristics; Chloride Ion; Chlorides; Computer Systems Development; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Development; Disruption; Engineering; Epithelial; Epithelium; Exhibits; Family suidae; Gene Transfer Techniques; Genes; Genotype; Human; Immunosuppression; In Vitro; Insertional Mutagenesis; Invasive; Lung; Lung diseases; Measurement; Modeling; Morbidity - disease rate; Mucous Membrane; Multipotent Stem Cells; Mus; Mutation; Outcome; Pharmacotherapy; Phenotype; Physiological; Property; Pulmonary Cystic Fibrosis; Pulmonary Pathology; RNA Interference; Regulation; Research Design; Resources; Stem cells; Structure; Study models; Sus scrofa; Techniques; Therapeutic; Therapeutic immunosuppression; Tissues; Transplantation; Weaning; desire; disease phenotype; gene therapy; homologous recombination; human disease; in vivo; model development; mortality; novel strategies; nuclear transfer; research study

Pulmonary disease is a hallmark of cystic fibrosis (CF) and the most frequent reason for morbidity and mortality. Murine models of CF produced by targeted disruption of the CFTR gene or insertional mutagenesis have been successful in yielding the desired genotype, but significant pulmonary pathology has not been a phenotypic outcome associated with CFTR deficiency in these animals. A pig exhibiting a CF phenotype would provide an attractive model for the study and treatment of CF lung disease. In contrast to mice, the porcine lung shares many anatomical characteristics with humans including similarities in bronchial branching, vascular distribution and structure of the airway mucosa. Because of these similarities, porcine lungs have been used to replicate other pulmonary disease states found in humans and have served as models for the development of transplantation techniques and strategies for immunosuppression. The promise of gene therapy as a means to treat CF has not been realized in part because of the lack of a large animal model that exhibits phenotypic manifestations of CF comparable to that of humans. The central hypothesis of this proposal is that a CFTR deficient pig will exhibit a CF phenotype that closely resembles the human disease and provide an excellent model system for the development of therapeutic approaches for the treatment of CF. To address this hypothesis, our first objective will be to investigate characteristics of porcine CFTR regulation in a chloride secreting epithelium derived from the pig and compare the results to known properties of human CFTR. Results from these studies will provide new information on the regulation of CFTR channel function necessary for interpretation of data obtained from in vivo animal studies designed to track the progression of the CF disease phenotype. In the second objective, genetically modified pigs with impaired CFTR function will be produced by the introduction of the AF508 mutation by homologous recombination, taking advantage of porcine multipotent progenitor cells as a cellular resource for the nuclear transfer experiments. In addition, transgenesis with a transposon for regulated pCFTR RNA interference will also be explored as a means to control the level of CFTR expression. The successful development of this approach will yield important information on levels of CFTR expression necessary for normal secretory function, an issue critical for the development of effective gene therapy The third objective will identify specific in vitro and minimally invasive in vivo physiologic measurements to document the CF phenotype in clinically important epithelial tissues and to follow the progression of CF from post-weaned pigs to when they become sexually mature adults (within 6 months). The successful development of a porcine model for CF will be an important step toward the enhancement of pharmacotherapies and development of new approaches to gene therapy that can be used for the treatment of cystic fibrosis.

肺部疾病是囊性纤维化（CF）的标志，也是发病率和死亡率最常见的原因。通过靶向破坏CFTR基因或插入诱变产生的CF小鼠模型已成功产生所需的基因型，但在这些动物中，显著的肺部病理学并不是与CFTR缺陷相关的表型结果。表现出CF表型的猪将为CF肺病的研究和治疗提供有吸引力的模型。与小鼠相比，猪肺与人类具有许多解剖学特征，包括支气管分支、血管分布和气道粘膜结构的相似性。由于这些相似性，猪肺已被用于复制人类中发现的其他肺部疾病状态，并已作为移植技术和免疫抑制策略开发的模型。基因治疗作为治疗CF的手段的前景尚未实现，部分原因是缺乏表现出与人类相当的CF表型表现的大型动物模型。该提议的中心假设是CFTR缺陷型猪将表现出与人类疾病非常相似的CF表型，并为开发治疗CF的治疗方法提供了极好的模型系统。为了解决这一假设，我们的第一个目标将是调查的特点，猪CFTR的监管来源于猪的氯化物分泌上皮细胞，并比较结果的人CFTR的已知属性。这些研究的结果将提供CFTR通道功能调节的新信息，这些信息对于解释从旨在跟踪CF疾病表型进展的体内动物研究中获得的数据是必要的。在第二个目标中，将利用猪多能祖细胞作为核转移实验的细胞资源，通过同源重组引入AF 508突变来产生CFTR功能受损的遗传修饰猪。此外，还将探索用转座子进行转基因以调节pCFTR RNA干扰，作为控制CFTR表达水平的手段。这种方法的成功开发将产生关于正常分泌功能所必需的CFTR表达水平的重要信息，第三个目标是鉴定特异性体外和微创体内生理测量，以记录临床上重要的上皮组织中的CF表型，并跟踪CF从治疗后到治疗后的进展。断奶仔猪到性成熟的成年猪（6个月内）。CF猪模型的成功开发将是增强药物治疗和开发可用于治疗囊性纤维化的基因治疗新方法的重要一步。