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死性常染色体隐性遗传疾病，由囊性纤维化电导调节因子（CFTR）氯离子通道缺陷引起。尽管 CF 会影响多个器官，但肺部持续的细菌感染是该疾病最危及生命的部分。由于缺乏能够重现 CF 患者肺病自然进展的 CF 动物模型，CF 肺病治疗方法的开发受到阻碍。我们最近生成了一个 CFTR 敲除雪貂模型，该模型可以重现肺、胰腺、肝脏、肠道和输精管中的人类疾病表型。 CFTR 敲除雪貂表现出两种肺部定植表型，这将有助于开发针对新生儿早期 CF 快速致死性肺部细菌感染和导致 8 个月大死亡的缓慢进行性肺部细菌定植的疗法。我们寻求利用这种新模型开发重组腺相关病毒（rAAV）基因疗法来治疗CF肺病。需要解决的重要生物学问题包括：1) 人类和雪貂气道分泌物中发现的新型抑制蛋白的表征，该蛋白与 rAAV1 衣壳紧密结合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) 生成 rAAV-CFTR 载体，该载体能够逆转 CF 雪貂模型中的肺病，并与 rAAV 基因组的包装限制和有效的细胞要求相兼容。 CFTR 功能互补，以及 3) 识别肺部部位（气道表面上皮与粘膜下腺），必须针对这些部位进行有效补充 CF 肺部疾病。该提案的第三个目标利用了 Engelhardt 实验室快速生成克隆 CFTR 敲除雪貂的独特能力，这些雪貂在肺部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细胞生物学难题。该提案将显着增强该领域有效开发 CF 肺治疗策略的能力，不仅使用 rAAV 载体，还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.