EFFICACY AND SAFETY OF IN VIVO TRANSFER OF THE CFTR CDNA TE AIRWAY EPITHELIUM

CFTR CDNA TE 气道上皮体内移植的功效和安全性

• 批准号: 6242295
• 负责人: RONALD G CRYSTAL
• 金额: $ 9.04万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 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/6242295
• 关键词: Adenoviridae; athymic mouse; chimeric proteins; chloride channels; chlorine; complementary DNA; cystic fibrosis; fusion gene; gene expression; gene therapy; genetic promoter element; human genetic material tag; human subject; inflammation; ion transport; laboratory rat; recombinant DNA; reporter genes; respiratory epithelium; transfection /expression vector

The crux of gene therapy for cystic fibrosis is to transfer to the relevant airway epithelial cells an expression cassette containing the coding sequence of the normal human CFTR gene in a fashion that will re- establish normal CFTR function in the airway epithelium. Independent of the vector system used, there are three important elements to be transferred: the CFTR coding sequences, the 3' mRNA maturation signal, and the 5' promoter that supports the expression of these sequences. All available evidence suggests that the 5' promoter element appropriate to correct the airway epithelial CFTR deficiency state in an effective and safe fashion. In this context, the overall goal of this project is to identify promoters that will maximize the efficacy and safety of transfer of the human CFTR cDNA to the airway epithelium, in order to normalize CFTR function in the airways of individuals with cystic fibrosis on a chronic basis. The E1- replication deficient recombinant adenovirus with the promoters and relevant gene inserted in the E1 position will be used as the "model" vector in all studies. Among the classes of promoters and/or promoter elements to be characterized include: other constitutive viral promoters; 5 flanking regions of the normal CFTR gene; epithelial tissue specific promoters; and promotor elements that can be regulated by internal or exogenous stimuli relevant to enhancing efficacy and safety. Replication deficient adenovirus vectors will be constructed using promoters in each class driving the expression of a reporter gene or the normal human CFTR cDNA. These constructs will be used to define the relative amount of expression supported by promoters in each class in vitro and in vivo and characterize the ability of promoters in each class to support the expression of the normal human CFTR cDNA in a fashion sufficient to correct the deficiency of cAMP-inducible Cl- secretion in the airway epithelial cells from individuals with cystic fibrosis. These various classes of promoters in the adenovirus constructs will also be evaluated for their function in airway epithelium in the context of the inflammatory milieu of the airways in cystic fibrosis. Finally, based on the information derived, chimeric promoters will be constructed with features that will optimize the efficacy and safety of in vivo gene therapy for the respiratory manifestations of cystic fibrosis.

囊性纤维化基因治疗的关键是转移到 相关气道上皮细胞的表达盒，其含有 正常人CFTR基因的编码序列， 在气道上皮中建立正常的CFTR功能。独立于 在使用的载体系统中，有三个重要元素 转移：CFTR编码序列，3' mRNA成熟信号，和 支持这些序列表达的5 ′启动子。所有 现有的证据表明，5'启动子元件适合于 以有效的方式纠正气道上皮CFTR缺陷状态， 安全时尚在这方面，该项目的总体目标是 确定促进者，将最大限度地提高转让的效率和安全性 人CFTR cDNA的气道上皮细胞，以正常化 囊性纤维化患者气道中CFTR功能的研究 慢性基础E1复制缺陷型重组腺病毒， 将使用插入E1位置的启动子和相关基因 作为所有研究中的“模型”向量。在各类发起人中， 和/或启动子元件包括：其它组成型元件 病毒启动子;正常CFTR基因的5个侧翼区;上皮 组织特异性启动子;和可由 与增强功效和安全性相关的内部或外部刺激。 复制缺陷型腺病毒载体将使用 驱动报告基因表达的每个类别中的启动子或 正常人CFTR cDNA。这些构造将用于定义 每类中由启动子支持的表达的相对量， 体外和体内，并表征每类启动子的能力 以某种方式支持正常人CFTR cDNA的表达 足以纠正cAMP诱导的Cl-分泌的不足， 来自囊性纤维化患者的气道上皮细胞。这些 腺病毒构建体中的各种类型的启动子也将 评估它们在气道上皮中的功能， 囊性纤维化中气道的炎症环境。最后基于 将构建信息衍生的嵌合启动子， 优化体内基因治疗的功效和安全性的特征 治疗囊性纤维化的呼吸系统表现。