Core--Correction

核心--修正

基本信息

批准号：
7622055
负责人：
SHERIF E GABRIEL
金额：
$ 17.08万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7622055
关键词：
Affect Biological Assay Code Complement component C1s Confocal Microscopy Cyclic AMP Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Defect Disease Disease model Dose Effectiveness Epithelial Epithelial Cells Gastrointestinal tract structure Gene Transfer Hereditary Disease Human Hydration status In Vitro Inherited Ion Transport Ions Label Lead Liquid substance Lung Measurement Measures Mediating Microdialysis Mucous body substance Mus Nasal Epithelium Neonatal Nose Numbers Optics Organ Perinatal Preparation Property Proteins Pulmonary Cystic Fibrosis Rate Research Research Personnel Sampling Surface Techniques Testing Virus absorption airway surface liquid blue dextran concept cystic fibrosis mouse cytokine design epithelial Na+ channel gastrointestinal epithelium gene cloning gene therapy gene transfer vector in vivo monolayer mortality neonate prenatal vector

项目摘要

Cystic Fibrosis (CF) has been at the forefront of gene transfer research for the last decade. This hereditary monogenetic disease, although affecting epithelial cells of multiple organs, results most often in mortality due to complications associated with the lung. Cystic fibrosis lung disease has been considered as a prototypic disease state for "proof of concept" gene transfer strategies. The lack of an alternative long-term treatment for the pulmonary manifestations of this disease, the accessibility of the lung via the airway lumen, and the fact that viruses known to infect the lung were being developed into non-replicating gene transfer vectors led investigators to believe that administration of gene transfer vectors to the lung could potentially result in an effective treatment of this disease. CF is a disease of defective ion and fluid transport. The identification and cloning of the gene altered in CF demonstrated that this protein codes for a cAMP-mediated CI-channel, CFTR. It has also been demonstrated that defects in CFTR lead to decreased hydration of the airway surface liquid (ASL) and reduced mucus clearance. The assessment of the efficacy of gene transfer for CF requires a reliable assay to verify expression of the functional CFTR protein. In the Correction Core we will provide multiple techniques to detect the efficacy of CFTR gene transfer for correcting the ion and fluid transport defect of CF human and murine airway epithelial cells in vitro and CF murine nasal epithelia in vivo. We will use electrophysiological measurements across confluent monolayers mounted in Ussing chambers to determine the magnitude of both CI- (CFTR) and Na+ (ENaC) currents. Fluid transport rates will be assayed in the same preparations by confocal microscopy and absorption of blue dextran. To assess correction in vivo, we will measure nasal potential difference, the pH of ASL and the cytokine content collected by microdialysis. Gene transfer in the perinatal period may be most effective for treating a number of genetic diseases, including CF. We propose to study freshly excised epithelial sheets from prenatal and neonatal murine airways and gut. This will permit us to determine the effectiveness of in vivo vector dosing in neonatal CF (or other disease model) mice. The techniques available in this core will provide gene transfer investigators routine access to high-quality, high-sensitivity, well-controlled, robust measures of CFTR correction.

在过去的十年中，囊性纤维化（CF）一直处于基因转移研究的最前沿。这种遗传性的单基因疾病虽然影响了多个器官的上皮细胞，但由于与肺有关的并发症而导致死亡率最常导致。囊性纤维化肺疾病被认为是“概念证明”基因转移策略的原型疾病状态。缺乏对这种疾病的肺部表现，通过气道管腔的可及性以及已知感染肺的病毒的肺部表现的替代性治疗研究人员认为，将基因转移向量施用到肺部可能会导致对这种疾病的有效治疗。 CF是一种有缺陷的离子和液体转运的疾病。 CF中改变基因的鉴定和克隆表明，该蛋白质编码为CAMP介导的CI-通道CFTR。还已经证明，CFTR的缺陷导致气道表面液体（ASL）的水合减少和粘液清除率降低。基因转移对CF的疗效的评估需要可靠的测定来验证功能性CFTR蛋白的表达。在校正核心中，我们将提供多种技术来检测CFTR基因转移的功效，以纠正CF人和鼠气上皮细胞的离子和流体转运缺陷，并在体外和CF鼠鼻上皮中的含量体内。我们将使用安装在Ussing Chamber的汇合单层上的电生理测量值，以确定CI-（CFTR）和Na+（ENAC）电流的大小。通过共聚焦显微镜和蓝色葡萄糖的吸收，将在相同的制剂中测定流体传输速率。为了评估体内校正，我们将测量鼻电势差，ASL的pH和微透析收集的细胞因子含量。围产期基因转移可能最有效地治疗包括CF在内的多种遗传疾病。我们建议研究来自产前和新生儿鼠气道和肠道的新鲜切除的上皮床单。这将使我们能够确定新生儿CF（或其他疾病模型）小鼠中体内载体剂量的有效性。该核心中可用的技术将提供基因转移研究者的常规访问权限 CFTR校正。