Chitosan Increase The Efficient Of Adenovirus Vector Mediated Gene Transduction And Improve The Effect Of Oral Adenoviral-based Vaccines

Poor efficiency of adenoviral gene transfer to target cells is a major limitation to adenoviral gene therapy. Most inefficient gene transfer occurs in cells absence of coxsackie-and adenovirus receptor (CAR) on the cell surface. Chitosan, a well-characterized cationic polysaccharide, have been previously shown to enhance recombinant adenovirus vector infectivity in cells absence of CAR. However, its effect was only available in media of pH 6.4, but not physiological pH. Allowing for the insolubility of chitosan in physiological pH media may be responsible for that, we have developed a new method to dissolve chitosan in neutral and alkaline pH media with NaHCO3 solution. This chitosan solution was named chitosan sodium bicarbonate solution, and its physicochemical properties and cytotoxicity were investigated. An unexpected cytotoxicity of chitosan in pH6.4 media was found. The effect of this chitosan solution on adenovirus infectivity was evaluated in physiological pH media. The results clearly showed the chitosan sodium bicarbonate solution could enhance adenovirus infectivity in multiple cell lines in physiological PH media. In CHO B16 RD and DC2.4 cells which lack of adenovirus serotype 5 (Ad5) receptor, infectivity of adenovirus combination with chitosan was 8-24 fold greater than adenovirus alone. In A549 and Hep?cells which are sensitive to Ad5, chitosan also could enhance adenovirus infectivity slightly. This enhanced effect was chitosan concentration depended, and highest effect was obtained with 12.5?g-25?g/ml chitosan. We also investigated several influence factors on enhanced infectivity effect of chitosan solution, and found the effect of chitosan was stable in different circumstance. Our research provided a new procedure to dissolve chitosan in neutrality and alkaline pH media, and proved this solution could improve adenovirus infectivity on cells in physiological pH media, especially in cells without Ad5 receptor.After investigated the effect of chitosan sodium bicarbonate solution in vitro, we further investigated the application of this chitosan solution in vivo. We found the preparation of Mixing Chitosan solution with Pluronic F68 can greatly improve the tolerance of rAdv to simulated gastric acid fluid. and Trehalose can further improve the effect of Chitosan-Pluronic F68 Mixture. The result of gene transduction experiment indicated the Chitosan-Pluronic F68 Mixture can improve the efficiency of rAdv induced gene transduction in HT-29 cell line (human colon cancer cell line). Oral administration of rAdv-VP6 vaccine (adenovirus vector which expresses the VP6 protein of rotavirus) with Chitosan-Pluronic F68 Mixture could greatly increase seroconversion rates and serum titres of VP6 antibody which were remark higher than oral administration of rAdv-VP6 alone, and induced the mucosal IgA immunity to VP6 protein. After triple vaccination, the mice were challenged with 10DD50 EDIM (a mice strain of rotavirus) and the amount of rotavirus in mice stool was detected. The result shows oral administration of rAdv-VP6 vaccine with Chitosan-Pluronic F68 Mixture could markedly decrease the amount of rotavirus in mice stool, but oral administration of rAdv-VP6 vaccine had no effect. These result indicated the preparation of Chitosan-F68 could greatly increase immunity effect and protection effect of oral administration of rAdv-VP6.