MODIFYING TROPISM OF ADENOVIRUS VECTORS FOR GENE THERAPY

点击翻译按钮获取中文摘要

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.