A Ciliated Cell-Specific Promoter for Gene Therapy of CF

描述（由申请人提供）：本研究的长期目标 是开发一种纤毛细胞特异性启动子， 基因治疗或囊性纤维化（CF）的有效性。在正常的气道中， 囊性纤维化跨膜传导调节因子（CFTR）蛋白表达 主要在纤毛细胞的顶端表面和粘膜下腺体中。 为了使CF的基因治疗成功，正常的CFTR蛋白必须是 在适当的位置表达。然而，许多基因治疗载体 目前正在研究中，对分化的气道没有特异性 上皮此外，这些载体经常使用病毒启动子元件 或组成型表达基因的启动子来驱动高水平表达 报告基因。因此，使用这些载体的一个主要缺点是 它们可能导致不需要的细胞类型中CFTR的高水平表达 (e.g.,巨噬细胞、基底细胞）。这些启动子也可能不太有效， 在非分裂纤毛细胞中提供稳定、长期的表达 人口我们的假设是，使用特异性启动子来指导 CFTR蛋白表达于位于顶端的纤毛细胞 气道表面将纠正CF表型。另外我们 假设通过在整合载体中使用内源启动子， 将实现CFTR蛋白的稳定长期表达。的使用 纤毛细胞特异性启动子也将增加基因治疗的安全性 对于CF，通过防止CFTR在错误的基因中的潜在有害表达， 细胞类型。为了检验我们的假设，我们提出了以下具体目标： 具体目的1：鉴定并克隆纤毛蛋白基因启动子区 细胞特异性基因 具体目标2：确定负责 纤毛细胞特异性基因表达。 具体目的3：证明在体外两种情况下CF表型的校正 以及通过在纤毛细胞中靶向表达正常CFTR基因的体内模型， 细胞

DESCRIPTION (provided by applicant): The long-term objective of this research is to develop a ciliated cell-specific promoter that will improve the effectiveness of gene therapy or cystic fibrosis (CF). In normal airways, the cystic fibrosis transmembrane conductance regulator (CFTR) protein is expressed primarily at the apical surface of ciliated cells and in the submucosal glands. For gene therapy of CF to be successful, the normal CFTR protein must be expressed in the proper location. However, many of the gene therapy vectors currently under investigation have no specificity for the differentiated airway epithelium. In addition, these vectors frequently use viral promoter elements or promoters of constitutively expressed genes to drive high-level expression of reporter genes. A major drawback to the use of these vectors therefore is that they may result in high levels of CFTR expression in unwanted cell types (e.g., macrophages, basal cells). These promoters may also be less efficient at providing stable, long-term expression in the non-dividing ciliated cell population. Our hypothesis is that the use of a specific promoter to direct expression of the CFTR protein to the ciliated cells located at the apical surface of the airways will correct the CF phenotype. In addition, we hypothesize that by using an endogenous promoter in an integrating vector, we will achieve stable long-term expression of the CFTR protein. The use of a ciliated cell-specific promoter will also increase the safety of gene therapy for CF by preventing potentially deleterious expression of CFTR in the wrong cell types. To test our hypothesis, we propose the following specific aims: Specific Aim 1: To identify and clone the promoter regions of ciliated cell-specific genes. Specific Aim 2: To identify the essential regulatory elements responsible for ciliated cell specific gene expression. Specific Aim 3: To demonstrate correction of the CF phenotype in both in vitro and in vivo models by targeted expression of the normal CFTR gene in ciliated cells.