FVIIa as used pharmacologically is not TF dependent in hemophilia B mice.

Activated factor VII is approved for treating hemophilia patients with autoantibodies to their factor IX or FVIII; however, its mechanism of action remains controversial. Some studies suggest that FVIIa requires tissue factor (TF) for function and that the reason for the high dose requirement is that it must compete with endogenous FVII for tissue factor. Others suggest that FVIIa binds platelets where it activates FX directly; the high concentration required would result from FVIIa's weak affinity for phospholipids. We address this question by infusing a chimera of mouse FIX (Gla and EGF1) with FVIIa (EGF2 and catalytic domain) into hemophilia B mice. This mutant has no TF-dependent activity because it cannot functionally bind TF at physiologically relevant concentrations. In vivo, this mutant is as effective as mouse FVIIa in controlling bleeding in hemophilia B mice. Our results suggest that the hemostatic effect of pharmacologic doses of FVIIa is TF independent.

A 16bp Rep binding element is sufficient for mediating Rep-dependent integration into AAVS1.

Adeno-associated virus (AAV) is a non-pathogenic virus and the only known eukaryotic virus capable of targeting human chromosome 19 for integration at a well-characterized AAVS1 site. Its site-specific integration is mediated by Rep68 and Rep78, viral proteins that bind to both the viral genome and AAVS1 site on ch19 through a specific Rep-binding element (RBE) located in both the viral genome and AAVS1. There are three RBEs in the AAV genome: two identical ones in both inverted terminal repeats (ITR) and another one in a recently discovered region termed the P5 integration efficiency element (P5IEE) that encompasses the viral P5 promoter. In order to identify the viral cis-acting sequence essential for Rep-mediated integration, we tested a series of constructs containing various lengths of P5IEE and compared the two RBEs from ITR (RBE(itr)) and P5IEE (RBE(p5)) in terms of their efficiency in Rep-dependent integration. Methods employed included a colony-forming assay, a PCR-based assay and Southern blotting analysis. We found that 16bp of the RBE cis-element was sufficient for mediating Rep-dependent site-specific integration. Furthermore, RBE(itr) was both more effective and specific than the RBE(p5) in Rep-dependent integration at the AAVS1 site. These findings added new information on the mechanism of Rep-dependent AAV genome insertion at the AAVS1 site and may be helpful in developing new high efficiency vectors for site-specific transgene integration.

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.

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.

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.