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: 6109989
• 负责人: JOHN C OLSEN
• 金额: --
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109989
• 关键词: 3T3 cells; Macaca mulatta; Retroviridae; athymic mouse; butyrates; cell growth regulation; chloride channels; complementary DNA; cystic fibrosis; ferrets; gene expression; gene therapy; genetic manipulation; genetic regulatory element; genetic transduction; human genetic material tag; laboratory mouse; laboratory rat; mitogens; oxidizing agents; plasmids; receptor expression; respiratory epithelium; tissue /cell culture; transfection /expression vector; virus receptors; virus replication; xenotransplantation

The overall goal of this project is to maximize the potential of retrovirus vectors for gene transfer into the airway epithelium of cystic fibrosis (CF) patients. We will focus on the following aspects of gene transfer: (i) improving the efficiency of retrovirus entry into primary human epithelial cells; (ii) optimizing the persistence of transduced cystic fibrosis transmembrane conductance regulator (CFTR) gene expression in human airway epithelium; and (iii) developing methods to maximize gene transfer in vivo. The focus on retrovirus entry into airway epithelial cells involves (a) adaptation of retroviruses to replicate more efficiently these cells; and (b) localizing the GALV receptor in the airway epithelium and to determine how its expression changes during proliferative repair. The focus on transduced CFTR expression involves optimizing long-term expression in CF cells grown in culture and optimizing the persistence of expression during differentiation in vivo using the tracheal graft model. The emphasis on long-term expression is organized around the following functional themes: (a) the role of negative regulatory elements on long term expression; (b) the effect of CFTR over-expression on long term expression; (c) long-term expression using housekeeping promoters; and (d) testing a recently developed splicing vector. The focus on maximizing gene expression in vivo will be to: (a) maximize airway epithelium proliferation rates by exposure to inhaled oxidants coupled with exposure to mitogens; and (b) increase the duration of exposure to retrovirus vectors by the delivery of retrovirus packaging cell lines on micro-carrier beads. Our hypothesis is that retroviruses will be useful vehicles for gene transfer into the airways of newborn animals, where the proliferation rate is higher than adults, or alternatively, into the airways of adults whose epithelium is proliferating in response to limited injury, for example after exposure to oxidants. As a critical test of this hypothesis we will determine the efficiency and safety of retrovirus gene transfer to the airways using the rhesus monkey as a non-human primate model.

该项目的总体目标是最大限度地发挥 用于基因转移至囊性气道上皮的逆转录病毒载体 纤维化（CF）患者。 我们将重点关注基因的以下几个方面 转移：（i）提高逆转录病毒进入初级的效率 人上皮细胞； (ii) 优化转导的持久性 囊性纤维化跨膜电导调节基因（CFTR） 在人气道上皮中表达； (iii) 制定方法 最大化体内基因转移。 对逆转录病毒进入气道上皮细胞的关注涉及（a） 逆转录病毒的适应可以更有效地复制这些细胞；和 (b) 将 GALV 受体定位于气道上皮并 确定其表达在增殖修复过程中如何变化。 对转导 CFTR 表达的关注涉及优化长期 在培养物中生长的 CF 细胞中表达并优化持久性 使用气管移植物体内分化期间的表达 模型。 对长期表达的强调是围绕 以下功能主题：(a) 消极监管要素的作用 关于长期表达； (b) CFTR过度表达对长的影响 术语表达； (c) 使用持家启动子的长期表达； (d) 测试最近开发的剪接载体。 最大化体内基因表达的重点是：（a）最大化 暴露于吸入氧化剂导致的气道上皮增殖率 加上接触有丝分裂原； (b) 增加持续时间 通过逆转录病毒包装的递送暴露于逆转录病毒载体 微载体珠上的细胞系。 我们的假设是逆转录病毒将成为基因的有用载体 转移到新生动物的呼吸道，在那里增殖 率高于成人，或者进入成人呼吸道 其上皮细胞因有限损伤而增殖， 例如接触氧化剂后。 作为对此的关键测试 假设我们将确定逆转录病毒基因的效率和安全性 使用恒河猴作为非人类灵长类动物转移到呼吸道 模型。