RETROVIRUS-MEDIATED GENE TRANSFER INTO AIRWAY EPITHELIA

逆转录病毒介导的基因转移至气道上皮

• 批准号: 3366325
• 负责人: JOHN C OLSEN
• 金额: $ 19.62万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3366325
• 关键词: Retroviridae; beta galactosidase; cell differentiation; chlorine; clone cells; cystic fibrosis; cytokine receptors; electrophysiology; ferrets; forskolin; gene expression; gene therapy; genetic manipulation; genetic markers; genetic promoter element; genetic translation; histochemistry /cytochemistry; human genetic material tag; human subject; interleukin 2; ion transport; isoproterenol; laboratory rat; ozone; reporter genes; respiratory epithelium; sulfur oxides; tissue /cell culture; tissue /cell preparation; transfection; transposon /insertion element; virus virus interaction

Our goal is to develop a systematic approach to study genetic correction of the defect in cystic fibrosis lung disease. Specific goals include the following: First, retrovirus vectors containing the normal cDNA for the CFTR gene will be constructed and tested for their ability to mediate gene transfer into both human primary epithelial cells and CF cell lines. The stability of CFTR in these vectors will be evaluated. We will take advantage of preliminary results that have suggested specific vectors to use for efficient expression in epithelial cells. In addition, several approaches are proposed to increase the efficiency of retrovirus vector gene transfer, including the novel use of bicistronic vectors. A second goal is to investigate the airway biology pertinent to the introduction of foreign genes by retrovirus vectors. Primary human epithelium and immortalized cell lines will be used to study the efficiency of retrovirus gene transfer as well as the fate of vector expression during differentiation of airway epithelium. Also,, -.the types of cells that serve as targets for retrovirus vector infection will be determined. Selected experiments using ferrets will be used to study issues presently impossible to study in humans including: the correlation of retrovirus vector infection in vitro and in vivo; the persistence of vector expression in vivo; the effect of maturational factors on vector infection; and the effect of limited airway damage on the efficiency of retrovirus vector gene transfer. A third goal is to determine the fraction of CF airway cells in which the normal CFTR needs to be expressed in order for affected epithelia to attain normal bioelectric properties.

我们的目标是开发一种系统的方法来研究遗传 纠正囊性纤维化肺病的缺陷。 具体目标 包括以下内容： 首先，含有CFTR正常cDNA的逆转录病毒载体 将构建基因并测试其介导基因的能力 转移到人原代上皮细胞和 CF 细胞系中。 这 将评估 CFTR 在这些载体中的稳定性。 我们将采取 利用初步结果建议特定载体 用于在上皮细胞中有效表达。 此外，还有几个 提出了提高逆转录病毒载体效率的方法 基因转移，包括双顺反子载体的新用途。 第二个目标是研究与以下疾病相关的气道生物学： 通过逆转录病毒载体引入外源基因。 原初人类 上皮细胞和永生化细胞系将用于研究 逆转录病毒基因转移的效率以及载体的命运 气道上皮分化过程中的表达。 另外，-.the 作为逆转录病毒载体感染靶标的细胞类型将 被确定。 使用雪貂进行的选定实验将用于研究 目前无法在人类身上研究的问题包括： 逆转录病毒载体感染的体外和体内；的坚持 体内载体表达；成熟因素对载体的影响 感染;以及有限气道损伤对效率的影响 逆转录病毒载体基因转移。 第三个目标是确定 CF 气道细胞的比例 需要表达正常的 CFTR 才能受影响 上皮细胞获得正常的生物电特性。