1027. DNase II Knockdown in Human Endothelial Cells Does Not Improve Non-Viral Transfection Efficiency
Authors: Gregory A. Barker, Scott L. Diamond
Journal: Molecular Therapy (2006)

Endocytosis of non-viral vectors delivers them first to early endosomal compartments followed quickly by late endosomes. Vectors are then subject to possible sequestration to lysosomes. The acidic environment within lysosomes activates acidic nucleases which cause vector degradation. Deoxyribonuclease II (DNase II) is an acidic endonuclease most active at pH 4.5, which is primarily active in lysosomal compartments due to their low pH. DNase II has been identified as a lysosomal barrier to transfection, due to vector degradation upon lysosomal sequestration. However, the significance of this barrier on cationic gene transfer in nondividing endothelium has yet to be determined. Also, previous research has relied on techniques such as knock out models or chemical inhibitors, which could have non-specific effects on other proteins. RNA interference (RNAi) is a useful tool to knock down specific targets with control for off-targeting effects. Hence, to determine the significance of DNase II in nondividing human aortic endothelial cells (HAECs), we utilized RNA interference (RNAi) to knock down DNase II in vitro. We transfected HAECs at or near confluence with short-hairpin RNA (shRNA) targeted against human DNase II, using Lipofectamine 2000. DNase II knock down was verified using an acidic nuclease digestion gel assay. Cell lysates were incubated with lambda-phage DNA at pH 4.5 and run on an agarose gel to determine the relative nuclease activity. Several shRNA sequences targeting DNase II were transfected along with inactive shRNA controls. Comparison of these sequences revealed a shRNA sequence which achieved a significant knock down. Using the same gel based assay, we found the dose response of DNase II knock down was saturated above 75 nM. To determine the effect of DNase II knockdown on non-viral gene transfer, we transfected the shRNA followed by a transfection witha green fluorescent protein vector (pEGFP-N3) with Lipofectamine 2000. We verified that DNase II knock down remained in the presence of the plasmid using the acidic nuclease gel assay. The efficiency of the GFP plasmid delivery was quantified using flow cytometry. This data shows that shRNA-mediated DNase II knock down significantly decreases the transfection efficiency compared with untransfected controls. Additionally, the shRNA knock down provokes cytotoxicity and enhanced inflammatory IL-8 responses, which may cause the decreased transfection efficiency. Several short-interfering RNAs (siRNAs) were tested in a similar manner to determine if the decrease in transfection efficiency was sequence dependent. The siRNAs result in successful knock down of DNase II using the acidic nuclease assay. The siRNA-mediated knock down of DNase II results in transfection efficiencies about the same as the untransfected control. The cytotoxic effects on the cells is diminished following siRNA treatment, which further suggests that the shRNA sequence used causes deleterious effects. Therefore, these results demonstrate that DNase II is not a significant barrier to gene transfer in nondividing endothelium and the potential sequence- dependent effects of shRNA/siRNA on nonviral gene transfer.