rAAV-directed gene therapy to the lung in cystic fibrosis ferret models

描述（由申请方提供）：囊性纤维化（CF）是高加索人中最常见的致死性常染色体隐性遗传病，由囊性纤维化传导调节因子（CFTR）氯离子通道缺陷引起。虽然CF会影响多个器官，但肺部持续性细菌感染是该疾病最危及生命的组成部分。CF肺病的治疗的发展由于缺乏能够再现CF患者中肺病的自然进展的CF动物模型而受到阻碍。我们最近产生了一个CFTR基因敲除雪貂模型，该模型在肺、胰腺、肝、肠和输精管中再现了人类疾病表型。CFTR基因敲除雪貂表现出两种肺定植表型，这将有助于开发在新生儿早期期间肺的CF-快速致死性细菌感染的疗法，以及导致8个月龄死亡的肺的较慢进行性细菌定植。我们试图利用这种新的模型来开发CF肺病的重组腺相关病毒（rAAV）基因疗法。需要解决的重要生物学问题包括：1）鉴定在人和雪貂气道分泌物中发现的新的抑制性蛋白，其紧密结合rAAV 1衣壳并抑制体内基因转移，并剖析其细胞内蛋白酶体依赖性作用机制，2）rAAV的产生-CFTR载体能够逆转CF雪貂模型中的肺病，并且与rAAV基因组的包装限制和用于治疗的细胞要求相容。有效的CFTR功能互补，和3）鉴定肺中必须靶向CF肺病有效互补的位点（表面气道上皮对粘膜下腺体）。该提案的第三个目标是利用Engelhardt实验室的独特能力，快速产生克隆的CFTR敲除雪貂，这些雪貂在肺的生物相关细胞位点转基因表达重组fCFTR。因此，该提案解决了与改善气道基因治疗相关的新型病毒和细胞机制，同时还解决了有关CF肺病发病机制和治疗的困难细胞生物学问题。该提议将显著增强该领域有效开发用于治疗CF肺的治疗策略的能力，不仅使用rAAV载体，而且使用其他药理学和基于基因的疗法。

DESCRIPTION (provided by applicant): Cystic Fibrosis (CF) is the most common lethal autosomal recessive disease in Caucasians and is caused by defects in the cystic fibrosis conductance regulator (CFTR) chloride channel. Although multiple organs are affected in CF, persistent bacterial infections in the lung are the most life-threatening component of the disease. The development of treatments for CF lung disease has been hindered by the lack of CF animal models capable of reproducing the natural progression of lung disease in CF patients. We recently generated a CFTR-knockout ferret model that reproduces human disease phenotypes in the lung, pancreas, liver, intestine, and vas deferens. The CFTR-knockout ferrets demonstrate two lung colonization phenotypes that will be useful in developing therapies for CF-rapidly lethal bacterial infections of the lung during the during the early neonatal period and a slower progressive bacterial colonization of the lung that leads to death by 8 months of age. We seek to use this new model to develop recombinant adeno-associated virus (rAAV) gene therapies for CF lung disease. Important biologic problems to be addressed include: 1) characterization of a novel inhibitory protein found in human and ferret airway secretions, which tightly binds to the rAAV1 capsid and inhibits in vivo gene transfer, and dissection of its intracellular proteasome-dependent mechanisms of action, 2) the generation of rAAV-CFTR vectors that are capable of reversing lung disease in the CF ferret model and compatible with both packaging limitations of the rAAV genome and cellular requirements for efficient CFTR functional complementation, and 3) identification of the sites in the lung (surface airway epithelium vs submucosal glands) that must be targeted for effective complementation of CF lung disease. The third goal of the proposal draws on the unique ability of the Engelhardt laboratory to rapidly generate cloned CFTR-knockout ferrets that transgenically express recombinant fCFTR at biologically relevant cellular sites the lung. Thus, this proposal addresses novel viral and cellular mechanisms that are relevant to improving gene therapies to the airway, while also tackling difficult cell biology questions about the pathogenesis and treatment of CF lung disease. This proposal will significantly enhance the field's ability to effectively develop therapeutic strategies for treatment of the CF lung, not only using rAAV vectors, but also other pharmacologic and gene-based therapies.