PROTAMINE ENHANCES LIPOSOME-MEDIATED GENE TRANSFER EFFICIENCY TO HUH7 CELL LINE

Abstract

182 Liposome-mediated ex vivo gene transfer is a well established method for hepatocyte gene therapy. However, there is still room to improve its gene transfer efficiency. Protamine sulfate, which had already been applied clinically as an antidote for heparin, are small basic proteins with a high arginine content and is potent in folding the DNA. DNA-protamine complex may acquire a very condensed form. In this compact structure, DNA is well protected from enzymatic hydrolysis and may reach the nucleus in an intact form. Thus it may prevent itself from the digestion by the intracellular enzymatic hydrolysis and make gene transfer efficient. The reporter gene used here is green fluorescent protein (GFP). A simplified gel retardation assay was done to determine the minimal required amount of protamine to bind 5 μg of GFP plasmid and then various amounts of protamine sulfate (0, 10, 50, 100, 500 μg) were added into the foreign DNA-liposome mixture and transfected into 6-well Huh7 cell culture plate with the density of 6×105 cells per well. Expression index (EI), the ratio of the percentage of cells which could emit the green fluorescence between the variable experimental groups and the lipofectamine vector group was investigated by FACScan cell counting. All experiments were quadriplicated. The results showed the expression index in 10 μg group: 3.4±2.3 (p<0.05), 50 μg group: 2.1±0.3 (p<0.05), 100 μg group: 1.9±0.7(p<0.05), and 500 μg 1.1±1.0 (p>0.05) when compared with the lipofecatmine group (EI=1). Based upon our findings, protamine sulfate did increase the gene expression efficiency of GFP in Huh7, especially at the dose of 10 μg for 5μg of GFP gene in 6×105 cells for transfection. It is very promising for this drug to apply in ex vivo hepatocyte gene therapy and it is also applicable in vivo if it can be modified to carry the foreign gene into hepatocytes.