Optimization of transfection mediated by calcium phosphate for plasmid rAAV-LacZ (recombinant adeno-associated virus-beta-galactosidase reporter gene) production in suspension-cultured HEK-293 (human embryonic kidney 293) cells

摘要

rAAV (recombinant adeno-associated virus) has become a very useful gene-delivery vector for gene there apy. However, it is very difficult to generate rAAV using triple transfection on a commercial scale, owing to its low productivity and inconveniently adhesive nature of its culture. An optimal suspension-culture transfection procedure was developed for rAAV-LacZ production in suspended HEK-293 cells mediated by calcium phosphate (IacZ, a reporter gene, codes for beta-galactosidase). The study showed that cytotoxicity of transfection complexes and cell aggregation in suspension culture were two key factors affecting high suspension-culture transfection efficiency. Cytotoxicity of transfection complexes was influenced effectively by mixture of Ca2+ and plasmid DNA when their concentrations were decreased from 300 to 150 mM and from 3.0 to 1.5 mu g/ml respectively, as manifested by a relatively higher cell viability after suspension-culture transfection. Moreover, the transfection efficiency was still less than 15%. In addition, we explored the disruption of cell aggregation and the control of transfectioncomplex size with 2.0 mM EGTA treatment for 30 min before transfection and the addition of 100 mM Mg2+ during transfection respectively, procedures which enhanced transfection efficiency significantly, owing to more contact and endocytosis between cells and transfection complexes. Finally, the high transfection level and rAAV-LacZ titre achieved under optimized suspension-culture transfection conditions, namely 40% and 5 x 10" v.g. (vector genomes)/60 ml of medium respectively, is promising for the technique's application in the large-scale production of rAAV.