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

描述(申请人提供)：囊性纤维化(CF)是高加索人最常见的致命性常染色体隐性遗传病，由囊性纤维化传导调节因子(CFTR)氯通道缺陷引起。尽管多个器官都会受到影响，但肺部持续的细菌感染是这种疾病最危险的组成部分。由于缺乏能够复制CF患者肺部疾病自然进展的CF动物模型，CF肺部疾病的治疗方法的发展一直受到阻碍。我们最近建立了一种CFTR基因敲除雪貂模型，该模型在肺、胰腺、肝脏、肠道和输精管中复制了人类疾病的表型。CFTR基因敲除雪貂展示了两种肺部定植表型，这两种表型将有助于开发治疗CF的方法-新生儿早期肺部快速致死性细菌感染和导致8个月大前死亡的较慢的肺部细菌定植。我们试图利用这一新的模型来发展重组腺相关病毒(RAAV)基因治疗慢性肺病。需要解决的重要生物学问题包括：1)鉴定在人和雪貂呼吸道分泌物中发现的一种新的抑制蛋白，它与rAAV1衣壳紧密结合并在体内抑制基因转移，并剖析其细胞内依赖蛋白酶体的作用机制；2)构建能够逆转CF雪貂模型中的肺部疾病的rAAV-CFTR载体，并且与rAAV基因组的包装限制和有效的CFTR功能互补的细胞要求相兼容；以及3)确定有效补充CF疾病必须针对肺组织的位置(表面呼吸道上皮与粘膜下腺)。该提案的第三个目标利用了恩格尔哈特实验室的独特能力，即快速培育克隆的CFTR基因敲除雪貂，在肺的生物相关细胞部位转基因表达重组fCFTR。因此，这项建议解决了与改进对呼吸道的基因治疗相关的新的病毒和细胞机制，同时也解决了关于CF肺部疾病的发病机制和治疗的困难的细胞生物学问题。这项建议将显著提高该领域有效开发治疗策略治疗CF肺的能力，不仅使用rAAV载体，而且还使用其他药物和基于基因的治疗方法。

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.