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）肺部疾病导致呼吸道上皮表面衬里脱水，导致粘膜淤积和慢性细菌感染。 CF 中缺失的基因产物是囊性纤维化跨膜电导调节蛋白 (CFTR)，它调节气道上皮中的液体稳态机制。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。 CF肺病的基因转移策略迄今为止未能显示出显着的CFTR在体内递送至气道细胞，这主要是因为载体在管腔内递送后未能有效地将CFTR转移至呼吸道上皮。我们发现重组副流感病毒（rPIV）在管腔内递送后可高效感染人分化良好的气道细胞（HAE）培养物，并且基因转移对纤毛气道上皮细胞具有特异性，而纤毛气道上皮细胞是CF中需要校正的细胞类型。 rPIV 向纤毛细胞的基因转移效率高、PIV 重组版本的可用性以及将大型转基因插入 rPIV 基因组的能力表明这些病毒可能有助于将 CFTR 传递至 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，我们建议生成表达人类CFTR的新型重组rPIV基因转移载体，并测试这些载体在纠正CF HAE的CF表型方面的功效和安全性。我们还将在非人灵长类动物的鼻上皮中测试 rPIV 载体的效率、安全性以及免疫系统对基因转移的影响。拟议研究的具体目标是： 1) 能否生成 rPIV 载体以将 CFTR 基因有效且安全地转移至纤毛气道细胞？ 2) 体外测试rPIV-CFTR载体对人高分化气道上皮细胞的功效和细胞毒性； 3) 测试 rPIV 载体在非人灵长类动物鼻上皮体内的基因转移效率。