CFTR Delivery to Ciliated Airway Cells by PIV Vectors

通过 PIV 载体将 CFTR 递送至纤毛气道细胞

• 批准号: 6970150
• 负责人: RAYMOND J PICKLES
• 金额: $ 36.33万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-06 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6970150
• 关键词: Macaca mulatta; Paramyxovirus; biotechnology; chloride channels; clinical research; cystic fibrosis; densitometry; gene delivery system; gene expression; gene targeting; gene therapy; genetic transcription; green fluorescent proteins; human tissue; immunofluorescence technique; microarray technology; molecular cloning; nonhuman therapy evaluation; polymerase chain reaction; recombinant virus; respiratory disease /disorder therapy; respiratory epithelium; therapy adverse effect; transfection /expression vector; western blottings

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.

描述（由申请人提供）：囊性纤维化（CF）肺部疾病会导致呼吸性上皮的表面内衬脱水，导致粘膜和慢性细菌感染。 CF中的缺乏基因产物是囊性纤维化跨膜电导调节剂（CFTR）蛋白，可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，从而恢复正常的肺功能。到目前为止，CF肺疾病的基因转移策略未能显示出体内的CFTR向气道细胞的显着递送，这主要是因为载体在内递送后没有有效地将CFTR转移到呼吸道上皮。我们发现，重组副氟氟扎病毒（RPIV），在腔内递送后具有高效率的人类良好分化的气道细胞（HAE）的培养，并且该基因转移特定于纤毛的气道上皮细胞，该细胞需要校正。 RPIV向纤毛细胞的基因转移效率很高，PIV的重组版本的可用性以及将大型转基因插入RPIV基因组中的能力表明，这些病毒可能有助于将CFTR传递到CF肺的纤毛纤维中的Airway上皮。但是，目前可用的PIV载体是纤毛细胞的细胞毒性。因此，我们建议生成新型的基于RPIV的基因转移载体，以表达人CFTR并测试这些载体在校正CF HAE的CF表型时的功效和安全性。我们还将测试免疫系统在体内非人类灵长类动物鼻上皮中RPIV媒介中免疫系统对基因转移的效率，安全性和影响。拟议的研究的具体目的是：1）可以生成RPIV向量以有效且安全的CFTR基因转移到纤毛气道细胞中吗？ 2）测试RPIV-CFTR载体对人体差异良好的气道上皮细胞的疗效和细胞毒性； 3）测试在体内非人类灵长类动物鼻上皮中RPIV向量的基因转移效率。