MODIFYING TROPISM OF ADENOVIRUS VECTORS FOR GENE THERAPY

描述(直接从应用程序中获取) 囊性纤维化是一种常染色体隐性遗传性疾病，由 囊性纤维化跨膜电导调节因子的突变 吉恩。以前使用腺病毒载体的基因治疗方法已经 有两个主要缺点：(1)需要使用大剂量的病毒 由于腺病毒效率较低而转导cftr基因 进入实际肺组织和(2)引起的非特异性炎症 病毒。在此应用程序中，我们建议测试新的假设 添加到纤维蛋白C-末端的配体可能会限制进入 转化成特定的细胞子集，并可能提高载体的效率 进入这些牢房。我们已经证明了另一个细胞的新配体 表面受体可以与腺病毒的羧基末端融合。 纤维蛋白，它在感染过程中介导最初的进入步骤，以及 这些配体可以被针对该配体的抗体所访问。我们 提出三个具体目标，重点是提高企业的准入效率 腺病毒载体：(1)腺病毒中的哪些结构域决定 形成三聚体的能力和与正常细胞表面结合的能力 受体？我们将继续绘制影响纤维的突变图谱 形成三聚体的能力，将使用独特的单抗进行检测 只识别三聚体的抗体。我们将进行替换突变 在从纤维蛋白延伸出来的五个环中的每一个中，测试是否 新的表位可以通过插入到这些环中来添加，如果中和 表位可以删除。(2)细胞的进入途径和细胞趋向性能否 腺病毒载体可通过添加额外的单一表位进行修饰 放在纤维旋钮上？我们将使用蛋白质和病毒结合来识别 阻止与正常受体结合的位点，并询问我们的 “连接”的纤维能够结合正常的细胞受体和/或 由添加的配基指定的受体。(3)使用模型系统的重要性 对于囊性纤维化，我们能否显示出特异性的细胞附着和进入 趋向性修饰腺病毒载体？我们将使用组织培养细胞， 小鼠气管环，和小鼠气管判断是否特异 添加到纤维上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 细胞类型和/或阻断通过正常病毒受体的载体进入。

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.