Directed evolutioni of AAV for gene theraphy in a pig model of cystic fibrosis

囊性纤维化（CF）是一种由囊性纤维化跨膜传导调节因子（CFTR）缺失引起的遗传性疾病。该疾病涉及许多器官，肺部疾病是目前发病率和死亡率的主要原因。基因转移提供了在患者肺部表达CFTR的潜力，从而减缓或预防疾病进展。然而，尽管取得了显著的研究进展，我们仍然缺乏许多关键问题的答案。进展的一个主要障碍是缺乏一种动物模型来复制通常在人类身上发现的疾病。为了规避当前动物模型的局限性，我们生产了CFTR-/-和CFTRAF/-猪。新生动物表现出CI转运缺陷，并在新生人类中复制异常。我们的初步数据表明，CFTR-/-猪也可能像人类一样发生呼吸道疾病。我们将使用这个新颖的模型来解决CF的关键问题。

Cystic fibrosis (CF) is a genetic disease caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR). The disease involves many organs, and lung disease is the current major cause of morbidity and mortality. Gene transfer offers the potential to express CFTR in the lungs of patients and thereby slow or prevent disease progression. Yet despite outstanding research progress, we still lack answers to many crucial questions. A major impediment to progress has been the lack of an animal model that replicates disease typically found in humans. To circumvent limitations of current animal models, we have produced CFTR-/- and CFTRAF/- pigs. Newborn animals exhibit defective CI' transport and replicate abnormalities in newborn humans. Our preliminary data suggest that CFTR-/- pigs may also develop respiratory disease like humans. We will use this novel model to address questions key to CF. Aim 1. Will intestinal expression of CFTR prevent meconium ileus in CFTRAF508/- pigs? To answer this question, we will generate CFTRAF508/- pigs carrying a transgene expressing CFTR in the intestine. The resulting animals may be of value to other projects in the program, and the results may shed light on the underlying pathogenesis of meconium ileus and distal intestinal obstruction syndrome. Aim 2. Do genetic modifiers influence the clinical phenotype of CFTRAF508/AF508 pigs? We will cross our pigs to genetically diverse strains of pigs and ask how the CF phenotypes are altered. Discovering phenotypic changes could help identify pathways that modify the AF508 mutant protein or affect manifestations of the disease, thereby revealing novel therapeutic targets. Aim 3. When is CFTR expression required to alter the CF phenotype? We will generate CFTRAF508 pigs that express CFTR under control of an inducible promoter to answer key questions. Will CFTR expression in utero prevent disease in newborns? Will epithelial CFTR expression that begins after birth prevent lung disease? Will CFTR expression treat or slow the progression of established ainway disease? The answers will serve as a benchmark to guide therapeutic strategies including gene transfer. RELEVANCE (See instructions): Research in cystic fibrosis (CF) has been impeded by lack of an animal model that replicates the typical manifestations of the disease. Our Program recently developed a porcine model of CF. We will use this novel model to better understand CF and hopefully accelerate the discovery and testing of gene transfer and other novel preventions and treatments.