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

囊性纤维化是由于囊性纤维化跨膜传导调节因子(CFTR)缺失引起的一种遗传性疾病。该病累及多个器官，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的肺部表达CFTR的可能性，从而减缓或防止疾病的进展。然而，尽管取得了显著的研究进展，我们仍然缺乏对许多关键问题的答案。进展的一个主要障碍是缺乏复制通常在人类中发现的疾病的动物模型。为了克服目前动物模型的局限性，我们培育了CFTR-/-和CFTRAF/-猪。新生动物在新生人类中表现出CI运输缺陷和复制异常。我们的初步数据表明，CFTR-/-猪也可能像人类一样患上呼吸道疾病。我们将使用这个新的模型来解决关键的CF问题。 目的1.CFTRAF508/-猪肠道表达CFTRAF508能预防胎粪肠梗阻吗？为了回答这个问题，我们将产生CFTRAF508/-在肠道中携带表达CFTR的转基因猪。由此产生的动物可能对该计划的其他项目有价值，结果可能有助于阐明胎粪性肠梗阻和远端肠梗阻综合征的潜在发病机制。目的2.遗传修饰物是否影响CFTRAF508/AF508猪的临床表型？我们将把我们的猪与遗传多样性的猪品系杂交，并询问CF表型是如何改变的。发现表型变化有助于确定修改AF508突变蛋白或影响疾病表现的途径，从而揭示新的治疗靶点。目的3.什么时候CFTR的表达才能改变CF的表型？我们将产生CFTRAF508头猪，在可诱导启动子的控制下表达CFTR，以回答关键问题。CFTR在子宫中的表达能预防新生儿的疾病吗？出生后开始的上皮性CFTR表达能预防肺部疾病吗？Cftr基因的表达能否治疗或延缓已建立的主干疾病的进展？这些答案将作为基准，指导包括基因转移在内的治疗策略。 相关性(请参阅说明)： 由于缺乏复制囊性纤维化的动物模型，囊性纤维化的研究一直受到阻碍。 该病的典型表现。我们的项目最近开发了一种猪的CF模型。我们将使用这个新的模型来更好地理解CF，并有望加快发现和测试基因转移和其他新的预防和治疗方法。

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.