Construction And Evaluation Of Adenovirus Vector Retargeting To Tumor Vessel

Background Along with the conventional therapeutic measures such as operation,radiotherapy and chemotherapy, gene therapy is now one of the most rapidly progressingfields in disease management. Now we have good reason to believe that gene therapywould become another powerful measures against disease. Currently, the main factorrestricting its application is lack of effective therapic genes and targetingvectors.The most commonly used gene delivery methods include: physical method,chemical methods, fusion method and gene gun etc. Despite their advantages, they oftenfail to achieve a high efficiency, and as a result, it's difficult to get the stable cell linesexpressing the interesting genes. The adenovirus vector is an excellent gene deliveryvector which has several key attributes.including: a broad host spectrum; ease ofproduction to high titer; high efficiency of infection; high level of gene expression;infection of both dividing and nondividing cells; no integration into the genome of thehosts; long fragment of insertion. All these features make the adenovirus an intriguingvector. However the adenovirus has no specificity of infection, therefore it is difficultto restrict the infection to the lesion.and some cell lines such as vascular endothelialcells, smooth muscular cells and hematopoietic cells are naturally resistant to adenovirusinfection, which greatly limits the application of adenovirus vector in genetherapy. Fiber protein of the adenovirus plays an important role in the adhesion ofadenoviruses to their host cells. By modifying the fiber protein, it's possible to renderadenovirus the specificity of infection, enrich its affinity and increase the efficiency ofgene delivery. Angiogenesis is a key factor in tumor growth, progress, metastasis andtherefore prognosis. Tumor vascular endothelial cells are normal endothelial cells, andthere is no significant difference between various types of tumor vascular. However thegene expression profiles of the tumor vascular endothelial cells and the normal vascularendothelial cells are different, which lays the premise of targeting the tumor vessels fortherapy. As mentioned above, the adenovirus can not infect the vascular endothelial cells,so the adenovirus must be modified before it can be used in targeting the tumor vessels.Objective Clone the full length of adenovirus fiber gene for the construction oftargeting adenovirus vector; modify the knob domain to construct the mutants of thefiber and evaluate the binding specificity of the mutants to the vascular endothelialcells;introduce the fiber mutants that can bind to the endothelial cells into the adenovirusgenome, pack the recombinant viruses in their host cells, investigate their ability ofbinding to the vascular endothelial cells and the efficiency of infection.Method (1) Clone the adenovirus fiber gene by endonuclease digestion, construct its prokaryotic and eukaryotic expression vector, analyze the structure of the fiber protein by denatured and non-denatured SDS-PAGE and western blot; (2) Introduce the EcoR V site into the knob domain of the fiber by site-directed mutagenesis, then insert the NGR motif into the EcoR. V site to get the mutant F.NGR, and then construct the prokaryotic and eukaryotic expression vector of the F.NGR mutant. The protein expressed was analyzed by denatured and non-denatured SDS-PAGE and western blot; (3) Introduce point mutation at r408 in fiber and F.NGR gene, (the mutants were named F.408 and F.NGR408 respectively), and construct their expressions vectors. The proteins expressed were analyzed by denatured and non-denatured SDS-PAGE and western blot; (4) Purify the fiber and its mutant proteins by the TALONspin column; (5) Investigate the specificity of the fiber and its mutant proteins to bind to target cells by immunocytochemical techniques; (6)Construct fiber rescue vector by gene recombination;(7) Transduce both F.NGR and the rescue vector into the bacteria to allow the insert...