Engineering a CFTR Deficient Porcine Model of CF

肺部疾病是囊性纤维化（CF）的标志，也是最常见的发病和死亡原因。通过靶向破坏CFTR基因或插入突变产生的CF小鼠模型已经成功地产生了所需的基因型，但在这些动物中，显著的肺部病理并不是与CFTR缺乏相关的表型结果。表现出CF表型的猪将为CF肺病的研究和治疗提供一个有吸引力的模型。与小鼠相比，猪肺与人类有许多解剖学特征，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，猪肺已被用于复制在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发展的模型。基因治疗作为一种治疗CF的手段的前景尚未实现，部分原因是缺乏一种大型动物模型，显示出与人类相当的CF表型表现。本提案的中心假设是CFTR缺陷猪将表现出与人类疾病非常相似的CF表型，并为CF治疗方法的开发提供了一个极好的模型系统。为了解决这一假设，我们的第一个目标将是研究猪CFTR在来自猪的氯化物分泌上皮中的调节特征，并将结果与人类CFTR的已知特性进行比较。这些研究的结果将提供有关CFTR通道功能调控的新信息，这对于解释旨在追踪CF疾病表型进展的体内动物研究数据是必要的。在第二个目标中，利用猪多能祖细胞作为核移植实验的细胞资源，通过同源重组引入AF508突变，产生CFTR功能受损的转基因猪。此外，还将探索利用转座子介导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获得正常分泌功能所需的CFTR表达水平的重要信息。第三个目标将确定特定的体外和微创体内生理测量，以记录CF在临床重要上皮组织中的表型，并跟踪CF从断奶仔猪到性成熟成人（6个月内）的进展。CF猪模型的成功开发将是加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。

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.