CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Gene Delivery to the Rat Liver Using Cationic Lipid Emulsion/DNA Complex: Comparison between Intra-arterial, Intraportal and Intravenous Administration

OBJECTIVE: To compare the efficiency of intra-arterial, intraportal, and intravenous administration of cationic lipid emulsion/DNA complex, as used for gene transfer to rat liver. MATERIALS AND METHODS: DNA-carrier complex for the in-vivo experiment was prepared by mixing DNA and a cationic lipid emulsion. According to the administration route used (intra-arterial, intraportal, or intravenous), the animals were assigned to one of three groups. The heart, lung, liver, spleen and kidneys were removed and assayed for total protein and luciferase concentration. RESULTS: The cationic lipid emulsion/DNA complex used successfully transfected the various organs via the different administration routes employed. Luciferase activity in each organ of untreated animals was negligible. Liver luciferase values were significantly higher in the groups in which intra-arterial or intraportal administration was used. CONCLUSION: The intra-arterial or intraportal administration of cationic lipid emulsion/DNA complex is superior to intravenous administration and allows selective gene transfer to the liver.

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression in-creased as cation concentration of vehicle dec-reased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression in-creased as cation concentration of vehicle dec-reased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

Enhanced Expressions and Histological Characteristics of Intravenously Administered Plasmid DNA in Rat Lung

Cationic liposome-mediated gene transfection is a promising method for gene therapy. In this study, the transfection efficiency and histological patterns were evaluated in rat lung after intravenous administration via femoral vein of naked plasmid DNA, naked plasmid DNA with pretreatment of DOTAP, and DOTAP-cholesterol-plasmid DNA complex. Plasmid DNA encoding bacterial LacZ gene was used. For quantification of LacZ gene expression, -galactosidase assay was performed. For histologic examination, X-gal staining and immunohistochemical staining for transfected gene products were performed. Pretreatment of DOTAP prior to the infusion of naked plasmid DNA increased transfection efficiency up to a level comparable to DOTAP-cholesterol-plasmid DNA complex injection. Transfected genes were mainly expressed in type II pneumocytes and alveolar macrophages in all animals. We conclude that the high transfection efficiency is achievable by intravenous administration of naked plasmid DNA with pretreatment of DOTAP, to a level comparable to DOTAP-cholesterol-plasmid DNA complex. In this regard, naked plasmid DNA administration with pretreatment of DOTAP could be a more feasible option for intravenous gene transfer than DOTAP-cholesterol-plasmid DNA complex, in that the former is technically easier and more cost-effective than the latter with a comparable efficacy, in terms of intravenous gene delivery to the lung.