CFTR Delivery to Ciliated Airway Cells by PIV Vectors

描述（由申请人提供）：囊性纤维化（CF）肺部疾病引起呼吸道上皮表面衬里脱水，导致粘膜停滞和慢性细菌感染。CF中缺失的基因产物是囊性纤维化跨膜传导调节蛋白（CFTR），该蛋白调节气道上皮中的流体稳态机制。我们假设CF患者气道上皮中功能性CFTR的表达将恢复CFTR功能，从而恢复正常肺功能。到目前为止，CF肺病的基因转移策略未能在体内显示CFTR向气道细胞的显著传递，主要是因为载体在腔内传递后不能有效地将CFTR转移到呼吸道上皮。我们发现重组副流感病毒（rPIV）在腔内传递后能高效感染人高分化气道细胞（HAE）的培养物，并且基因转移是特异性的纤毛气道上皮细胞，纤毛气道上皮细胞是CF中需要纠正的细胞类型。将大的转基因插入rPIV基因组的能力表明，这些病毒可能有助于将CFTR传递到CF肺的纤毛气道上皮。然而，目前可用的PIV载体对纤毛细胞具有细胞毒性。因此，我们提出构建新的重组rpiv基因转移载体，表达人CFTR，并测试这些载体在纠正CF HAE的CF表型方面的有效性和安全性。我们还将在体内测试rpiv载体在非人灵长类动物鼻上皮中基因转移的有效性、安全性和免疫系统的影响。本研究的具体目的是：1)能否产生rpiv载体，将CFTR基因有效、安全地转移到纤毛气道细胞中？2)体外检测rPIV-CFTR载体对人高分化气道上皮细胞的作用及细胞毒性；3)检测rpiv载体在非人灵长类动物鼻上皮内的基因转移效率。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) lung disease causes dehydration of the surface lining of the respiratory epithelium leading to mucostasis and chronic bacterial infection. The absent gene product in CF is the cystic fibrosis transmembrane conductance regulator (CFTR) protein that regulates fluid homeostatic mechanisms in the airway epithelium. We hypothesize that expression of functional CFTR in the airway epithelium of CF patients will restore CFTR function and thus normal lung function. Gene transfer strategies for CF lung disease have so far failed to show significant CFTR delivery to airway cells in vivo, primarily because the vectors did not efficiently transfer CFTR to the respiratory epithelium after intraluminal delivery. We have found that recombinant parainfluenza viruses (rPIV), infect cultures of human well-differentiated airway cells (HAE) with high efficiency after intraluminal delivery, and that gene transfer is specific to ciliated airway epithelial cells, the cell type requiring correction in CF. The high efficiency of gene transfer by rPIV to ciliated cells, the availability of recombinant versions of PIV and, the capacity to insert large transgenes into the rPIV genome suggests that these viruses may be useful for delivering CFTR to the ciliated airway epithelium of the CF lung. However, currently available PIV vectors are cytotoxic to ciliated cells. Therefore, we propose to generate novel recombinant rPIV-based gene transfer vectors that express human CFTR and test the efficacy and safety of these vectors at correcting the CF phenotype of CF HAE. We will also test the efficency, safety and the influence of the immune system on gene transfer by rPIV-vectors in the nasal epithelium of non-human primates in vivo. The Specific Aims of the proposed study are: 1) Can rPIV-Vectors be Generated for Efficacious and Safe CFTR gene transfer to Ciliated Airway Cells? 2) To Test the Efficacy and Cytotoxicity of rPIV-CFTR vectors on Human Well-Differentiated Airway Epithelial Cells In Vitro; and, 3) To Test the Gene Transfer Efficiency of rPIV-Vectors in the Non-Human Primate Nasal Epithelium In Vivo.