MODIFYING TROPISM OF ADENOVIRUS VECTORS FOR GENE THERAPY

改变腺病毒载体的趋向性用于基因治疗

• 批准号: 6056419
• 负责人: JEFFREY ALLEN ENGLER
• 金额: $ 24.53万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6056419
• 关键词: Adenoviridae; cystic fibrosis; epitope mapping; gene therapy; human tissue; laboratory mouse; tissue /cell culture; transfection /expression vector; virus genetics; virus infection mechanism; virus receptors

DESCRIPTION (Taken directly from the application) Cystic fibrosis is an autosomal recessive hereditary disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previous gene therapy approaches using adenovirus vectors have suffered from two main drawbacks: (1) the need to use high doses of virus to transduce the CFTR gene because of the lower efficiency of adenovirus entry in actual lung tissue and (2) the non-specific inflammation induced by the virus. In this application, we propose to test the hypothesis that new ligands added onto the C-terminus of the fiber protein might restrict entry into a specific subset of cells and might improve the efficiency of vector entry into these cells. We have shown that new ligands for the other cell surface receptors can be fused to the carboxyl-terminus of the adenovirus fiber protein, which mediates the initial entry step during infection, and that these ligands are accessible to antibodies specific for the ligand. We propose three specific aims, focused on improving the efficiency of entry of adenovirus vectors: (1) What domains in the adenovirus determine the ability to form trimers and the ability to bind to the normal cell surface receptor? We will continue to map mutations in fiber that affect the ability to form trimers that will be detected using a unique monoclonal antibody that recognizes only trimers. We will make replacement mutations in each of the five loops that extend out from the fiber protein, to test if new epitopes can be added by insertion into these loops and if neutralizing epitopes can be deleted. (2) Can the entry pathways and cell tropism for adenovirus vectors be modified by addition of additional single epitopes onto the fiber knob? We will use protein and virus binding to identify sites which block binding to the normal receptor and ask whether our "liganded" fiber is capable of binding the normal cell receptor and/or the receptor specified by the added ligand. (3) Using model systems important to cystic fibrosis, can we demonstrate specific cell attachment and entry of tropism-modified adenovirus vectors? We will use tissue culture cells, mouse tracheal rings, and mouse trachea to determine whether specific ligands added to fiber can redirect the protein to new lung-specific receptors and/or allow more efficient entry of the virus vector into these cell types and/or block vector entry via the normal virus receptor.

描述（直接取自应用程序） 囊性纤维化是一种常染色体隐性遗传性疾病， 囊性纤维化跨膜传导调节因子（CFTR）突变 基因 先前使用腺病毒载体的基因治疗方法具有以下优点： 有两个主要缺点：（1）需要使用高剂量的病毒 由于腺病毒效率较低， 进入实际肺组织和（2）由 病毒 在本申请中，我们提出测试新的 添加到纤维蛋白C末端的配体可能会限制进入 进入特定的细胞亚群，并可能提高载体的效率， 进入这些细胞。 我们已经证明了另一种细胞的新配体 表面受体可以融合到腺病毒的羧基末端 纤维蛋白，其在感染期间介导初始进入步骤，以及 这些配体可以被对该配体有特异性的抗体所接近。 我们 提出三个具体目标，重点是提高进入效率， 腺病毒载体：（1）腺病毒中的哪些结构域决定了腺病毒的功能。 形成三聚体的能力和与正常细胞表面结合的能力 受体？ 我们将继续绘制纤维中的突变， 形成三聚体的能力，将使用独特的单克隆抗体 只识别三聚体的抗体。 我们将进行替换突变 在从纤维蛋白质延伸出来的五个环中的每一个中，以测试是否 新的表位可以通过插入这些环而添加 表位可以被删除。 (2)细胞的进入途径和细胞向性 腺病毒载体可以通过添加额外的单表位来修饰 光纤旋钮上吗 我们将使用蛋白质和病毒结合来识别 阻断与正常受体结合的位点， “配体化”纤维能够结合正常细胞受体和/或配体。 由添加的配体指定的受体。 (3)使用模型系统很重要 囊性纤维化，我们能证明特异性细胞附着和进入 向性修饰的腺病毒载体？ 我们将使用组织培养细胞， 小鼠气管环，并确定小鼠气管是否特异 添加到纤维中的配体可以将蛋白质重定向到新的肺特异性 受体和/或允许病毒载体更有效地进入这些受体 细胞类型和/或阻断载体通过正常病毒受体进入。