The Hydraulic Method Of Gene Transfer System In The Tail Vein To Establish And Applications

(1) Efficient expression of human factor IX cDNA in liver mediated by hydrodynamics-based plasmid administration and studies of conditions on the efficiency of transfectionHydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor IX(hFIX) cDNA in 2.2 ml Ringer's solution into mice within 7 seconds. The peak level of hFIX expression was 2.5u,g/ml in mouse plasma 1 day after administration. The hFIX cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3 to 10 days.Various conditions that affect hydrodynamics-based gene delivery into mouse liver in vivo were also studied, such as DNA dose, effect of second injection, volume of injection fluid, speed of injection, type of solution, physiological conditions of animals (anesthetized or not), sex and strain of animals for their effect on the levels of human factor IX expression from the pCMV-hFIX plasmid. Expression of hFIX was increased with the increase of plasmid dose, and decreased with the second injection. Injection of 2.5ml or 5s was most effective to deliver DNA into mouse liver. Specific conditions such as the use of Ringer's solution as an injection fluid increased the efficiency of expression. Expression of hFIX in male was higher than female. ICR mice were better than C57 strain in high expression. This study would be helpful for the application of hydrodynamics-based procedure in study of gene transfer.(2) Insulin expression in liver of diabetic mice mediated by hydrodynamics-based administrationTransfer and expression of insulin gene is an alternative strategy to improve glycemic control in type I diabetes in vivo. Hydrodynamics-based procedure was proved to be very efficient for naked DNA transfer to the mouse liver. The basalhepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resume of glycemic control in type I diabetic mice. The engineered insulin cDNA was inserted into plasmid vectors driven by CMV promoter, and was transferred into STZ induced diabetic mice by hydrodynamic procedure. The body weight of treated mice, insulin levels, immunohistology of the liver, and quantity of insulin mRNA in the liver were assayed to identify the improvement of hyperglycemic complication after plasmid administration. A transposon system was also used to prolong the insulin expression in the liver. After plasmid administration, the plasma insulin was significantly increased in the diabetic mice and the livers were insulin-positive by immunostaining. At the same time the hyperglycemic complication was improved, and the blood glucose levels of mice were reduced to normal. The glucose tolerance of the treated diabetic mice was improved and body weight loss was also ameliorated. The rapid tail vein injection did not cause any fetal result. Our results suggested that insulin gene could be efficiently transferred into the liver of the diabetic mice via rapid tail vein injection and resulted in high level of insulin expression. The basal hepatic insulin production mediated by hydrodynamics-based administration improved the glycemic control in type 1 diabetes dramatically and ameliorated the diabetic manifestation. The hydrodynamics-based administration was a convenient and efficient way in the study of gene therapy for type I diabetes mellitus.