Intracellular DNA release and elimination correlate poorly with transgene expression after non-viral transfection

The intracellular limiting steps in non-viral gene delivery are still unclear. The purpose of this study was to quantize intracellular DNA release and elimination kinetics after transfection with various non-viral carriers (calcium phosphate precipitates, branched poly(ethyleneimine), poly-L-lysine, DOTAP, DOTAP/DOPE) and to correlate these factors with transgene expression. Intracellular kinetics of DNA was determined by novel quantitative method based on qRT-PCR and DNase treatment. Intracellular elimination of DNA after calcium phosphate transfection was rapid (half-life of 0.37 h) whereas the amount of DNA in the cells was stable for at least 136 h after poly(ethyleneimine) mediated transfection. Intracellular elimination half-lives for DNA delivered by other carrier systems ranged from 9 to 27 h. Calcium phosphate precipitates are not able to protect DNA, which explains the short elimination half-life. In the case of other carriers DNA is after complex removal mostly carrier bound but after 24 h the major fraction of DNA is in the released or loosened state. Overall, neither total nor released amount of intracellular DNA correlates with the transgene expression.