Enhancement And Sustained Expression Of Naked FⅨ DNA In Mice And M13 Phage Mediated Gene Delivery Vector

(1)Development of nonviral naked DNA transfection is attractive since they have several advantages over viral-based vectors, However, the relatively low and transient nature of gene expression limited the application of naked DNA transfer system.In our study, systematically investigated the effect of chloroquine on exogenous gene expressing in mice by hydrodynamics-based naked plasmid adminstration was reported. Hydrodynamics-based naked DNA plasmid administration was performed by tail vein injection of 10 (g of pCMV- hFⅨ and chloroquine (100(m, 200(m, 500(m) in 2.2 ml of Ringer' solution within 6-7s, the level and stability of hFⅨ expression, liver damaged and toxicity were then examined. The maximum expression of hFⅨ level was 4.4±1.8 (g/ml at 8 hours after injection, 9.7±1.6 (g/ml at 24 hours only existed in 200μM chloroquine-treated animals, which is 3-4 fold higher than that of control (P<0.001). There is no significant difference observed among all the treated groups, 3 days later. Transaminase level and liver histological study showed the damage of liver was not related to chloroquine (P>0.05). Our results demonstrated that chloroquine can enhance and sustain exogenous gene expression in vivo without side effect under our experimental conditions. We also investigated the effect of Linear fragment DNA(LF DNA)(eliminating bacterial backbone),circular DNA (C DNA), linear DNA (L DNA),among the different promoter, double and single chain with ITR on the expression of human clotting factor Ⅸ (hFⅨ) in mice. Compared with C DNA and L DNA, 1day after tail vein injection, LF DNA-treated animals expressed hFⅨ concentration of 9809 ng/mL (n=6), which is 1.5-4 folds higher than C DNA and L DNA, respectively (p<0.05). The hFⅨ expression was dropped to 264ng/mL at 30 days and persisted for more than 258 days. A four-fold decrease in the serum level of both TNF-α and IL-12 as compared with the C DNA and L DNA (P<0.01). Using LF DNA by eliminating all the bacteria backbone resulted in enhanced and sustained hFⅨ gene expression in vivo, suggesting that the enhanced and sustained hFⅨ gene expression was related with reduced inflammatory response. We also injected LF DNA into hemophilia B mouse, the results showed that the bleeding symptoms in the treated mice were significantly alleviated till our experiment finished (about 30 days). LF DNA provides a promising approach and useful therapeutic strategy to improve naked DNA delivery.There were no effect among the different promoter, double and single chain with ITR on the expression of human clotting factor Ⅸ (hFⅨ) in mice. (2)Although filamentous phage have recently been demonstrated to transduce mammalian cells, there is a significant need to increase transduction efficiency. In this article, when recombinant M13 phage particles are constructed by displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide) on their surfaces and carrying mammalian marker genes as part of their genome (Tat-phage), such Tat-phage can be highly efficiently internalized into mammalian cells: BHK, CHO, Hela, COS-1, and lead to reporter gene expression. GFP expression in 10.08% COS-1 is detected by FACS. In contrast, recombinant phage displaying RGD peptide induces less than 0.01% marker gene expression, and wild phage cannot induce detectable GFP expression. The results demonstrate that Tat peptide can be used to construct the phage delivery system.