Construction Of Efficient Non-viral Gene Vectors To Recombinate Dendritic Cells For Cancer Immunotherapy

Objective:Construction of efficient non-viral gene transfer vectors for DC gene delivery. Characterization of physical and chemical properties of these novel non-viral vector, and evaluaton of transfection efficiency and safety of materials/DNA nanocomplex in DCs. Evaluation of DC gene vaccine immunotherapy effect against melanoma cells in vivo.Methods:1.Synthesis and characterization of different non-viral gene transfer materials; 2.Preparation and characterization of various material/DNA nanocomplexes by incubation. After tranfection on cell lines by different material/DNA nanocomplexes, Spermine-Dextran(SD) was selected as the relatively better non-toxic and efficient non-viral gene transfer material for DCs.3.Transfection efficiency, cytotoxicity and mechanisms of distribution in cytoplasm and nucleus were all investigated in dendritic cells (DC) by using SD/DNA nanocomplexes; 4.By using SD/DNA complex, DCs were co-transfected with plasmid DNA encoding tumor-associated antigen and chemokine. Then we studied the transferred DCs migration behavior in vitro and in vivo. Finally, we examined vaccine protection treament effect in mice beared melanoma solid tumor.Results:After incubation, Spermine-Dextran (SD) and plasmid DNA can form homogeneous spherical composite nanoparticle complexes. The incubated complex shows relatively high transfect efficiency and low cytotoxicity in DCs. Endocytosis results showed DCs uptook SD/DNA into cytoplasm through energy dependendent lipid-raft mediated endocytosis pathway. After 4 hours transfection, plasmid DNA can enter into the nucleus. The SD/CCR7 plasmid complex transfection of DCs transfected by SD/CCR7 plasmid complexes appeared high migration activity in vivo. In ex vivo experiments, DCs were co-transfected by plasmids encoding gp100, a melanoma antigen peptide, and CCR7, a chemokine receptor. It showed good anti-tumor therapeutic effect after vaccination of the engineered DCs, and the survival of tumor-bearing mice was increased largely.Conclusion:SD/DNA nano-complex is a novel efficient non-viral gene transfer vector with low cytotoxicity for transfection of DC. DCs engineered by these complexes have a good vaccine effect for tumor immunotherapy and have a good clinical therapy potential...