Eliminating bacteria backbone of naked DNA enhanced hFIX expression and reduced inflammatory response in mice.

To test the hypothesis that the persistent high level of transgene expression of linear DNA eliminating bacterial backbone (LDEBB) results from less cytokine induction in vivo. We systematically investigated the effect of circular DNA (C DNA), linear DNA (L DNA) and LDEBB on gene expression in mice by hydrodynamics-based plasmid administration, and then determined serum cytokine levels in mice by enzyme linked immunosorbent assay (ELISA). The expression of human clotting factor IX (hFIX) gene in mice treated with LDEBB, L DNA or C DNA reached a maximum 1-day after injection (9809, 6447, 2368 ng/mL), respectively. Thirty days after injection, hFIX concentrations dropped to baseline in mice treated with C DNA group, while L DNA group and LDEBB group decreased to 207 and 377 ng/mL, respectively, at the same time-point. Mice receiving LDEBB encoding hFIX expressed approximately 1.5 to 20-fold more serum hFIX than mice injected with L DNA and C DNA for a period of 8 months, respectively. However, mice receiving LDEBB are much less inflammatory than L DNA and C DNA as shown by a 4-fold reduction in serum levels of both TNF-alpha and IL-12. These results demonstrate that LDEBB is not silenced and is capable of expressing persistently high levels of transgene in vivo, which result from less cytokine induction in vivo. LDEBB provides a promising approach and useful therapeutic strategy to improve naked DNA delivery.

Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase.

The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.

Insulin expression in livers of diabetic mice mediated by hydrodynamics-based administration.

AIM: Transfer and expression of insulin gene in vivo are an alternative strategy to improve glycemic control in type 1 diabetes. Hydrodynamics-based procedure has been proved to be very efficient to transfer naked DNA to mouse livers. The basal hepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resumption of glycemic control in type 1 diabetic mice. METHODS: Engineered insulin cDNA was inserted into plasmid vectors under a CMV promoter, and transferred into STZ induced diabetic mice by hydrodynamic procedure. Glucose levels, 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. Sleeping Beauty, a transposon system, was also used to prolong the insulin expression in the liver. RESULTS: After plasmid administration, 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. The blood glucose levels of mice were reduced to normal. Glucose tolerance of the treated diabetic mice was improved. Body weight loss was also ameliorated. The rapid tail vein injection did not cause any fatal result. CONCLUSION: Our results suggested that insulin gene could be efficiently transferred into the livers of diabetic mice via rapid tail vein injection and it 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 diabetic syndromes. Hydrodynamics-based administration offers a simple and efficient way in the study of gene therapy for type 1 diabetes.

Conditions affecting hydrodynamics-based gene delivery into mouse liver in vivo.

1. It has been demonstrated that the hydrodynamics-based procedure has high efficiency to deliver foreign genes into the liver. The widespread use of this procedure in gene function studies and as a treatment option for liver and other organ diseases puts considerable importance on the investigation of various conditions that affect hydrodynamics-based gene delivery into mouse liver in vivo. 2. Various conditions, including the volume, speed and solution of the injection and the state, gender and strain of the animal were manipulated to evaluate their effect on the expression levels in mice of human factor IX (hFIX) 8 h after tail vein injection of the plasmid pCMV-hFIX. 3. It was found that an injection volume of 2-2.5 mL and an injection speed of 5-7 s were very effective in delivering DNA into the mouse liver. Using Ringer's solution as an injection fluid increased the efficiency of hFIX expression. 4. Anaesthetized mice expressed higher hFIX than conscious mice. Males expressed higher hFIX than females. The ICR mouse strain demonstrated higher expression of the foreign gene than did the C57 strain. 5. The effects of these specific factors on hFIX expression may be caused by variations in hydrostatic pressure, the degree of liver damage and liver size. 6. It can be concluded that there are optimal conditions for hFIX expression in the liver. This information may be helpful for the application of hydrodynamics-based procedures in the investigation of gene expression and gene therapy.