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

描述（由申请人提供）：囊性纤维化（CF）是白种人中最常见的致命性常染色体隐性遗传病，由囊性纤维化电导调节剂（CFTR）氯离子通道缺陷引起。尽管CF患者的多个器官受到影响，但肺部持续的细菌感染是该疾病最危及生命的部分。由于缺乏能够再现CF患者肺部疾病自然进展的CF动物模型，阻碍了CF肺病治疗方法的发展。我们最近建立了一个cftr敲除的雪貂模型，该模型在肺、胰腺、肝脏、肠道和输精管中再现了人类疾病的表型。cftr基因敲除的雪貂表现出两种肺部定植表型，这将有助于开发治疗新生儿早期肺部快速致死性细菌感染和导致8个月大时死亡的缓慢进进性肺部细菌定植的疗法。我们试图利用这一新模型开发重组腺相关病毒（rAAV）基因治疗CF肺病。需要解决的重要生物学问题包括：1)表征在人和雪貂气道分泌物中发现的一种与rAAV1衣壳紧密结合并抑制体内基因转移的新型抑制蛋白，并解剖其细胞内蛋白酶体依赖的作用机制；2)生成能够在CF雪貂模型中逆转肺部疾病的rAAV-CFTR载体，该载体既符合rAAV基因组的包装限制，又符合高效CFTR功能互补的细胞要求。3)确定肺中必须靶向的部位（气道表面上皮与粘膜下腺），以有效补充CF肺部疾病。该提案的第三个目标是利用Engelhardt实验室的独特能力，快速生成克隆的cftr敲除雪貂，这些雪貂在肺的生物学相关细胞部位转基因表达重组的fCFTR。因此，本研究提出了与改善气道基因治疗相关的新型病毒和细胞机制，同时也解决了CF肺病的发病机制和治疗方面的细胞生物学难题。该提案将显著提高该领域有效制定治疗CF肺的治疗策略的能力，不仅使用rAAV载体，而且还使用其他药物和基因治疗。

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.