A novel nonviral vector based on vesicular stomatitis virus.

Here we report a simple and efficient method for nonviral gene transfer using liposomes which have envelope protein of vesicular stomatitis virus (VSV) on their surface (VSV-liposomes). We prepared VSV-liposome by fusing simple liposomes with VSV particles. The density of VSV-liposome fusion products was intermediated between that of liposomes and that of VSV particles. Furthermore, VSV-liposome fusion products included both viral proteins and lipids from liposomes, and were confirmed to be fusion products, but not adsorptive products, by the resonance energy transfer fusion assay. To evaluate whether these particles can efficiently introduce their internal contents into the cytoplasm of mammalian cells, we examined the delivery of fragment A of diphtheria toxin (DTA) by VSV-liposomes into the cytoplasm of FL cells. We found that VSV-liposomes encapsulating DTA were highly cytotoxic to the cells, while empty VSV-liposomes and plain liposomes encapsulating DTA were not, suggesting that VSV-liposomes delivered DTA into cytoplasm. Consistent with this, the cells cultured with plasmid DNA entrapped in VSV-liposomes and coding for firefly luciferase showed significant luciferase expression, whereas cells culture with plasmid DNA in plain liposomes and plasmid DNA-cationic liposomes complex did not. Thus, VSV-liposomes function as a simple and efficient nonviral vector for the delivery of DNA.

A novel vaccine delivery system using immunopotentiating fusogenic liposomes.

We previously reported the preparation and characterization of fusogenic liposomes (FLs), which have two highly immunogenic glycoproteins of the Sendai virus on their surface. In this report, we investigated the capacity of FLs to enhance antigen-specific humoral immunity in mice. FLs function as a lymphocyte mitogen with high immunogenicity consistent with viral envelope proteins. Markedly increased levels of anti-ovalbumin (OVA) antibody were detected in serum from mice immunized with OVA encapsulated in FLs compared to sera from mice immunized with free OVA or OVA encapsulated in plain liposomes. An anti-OVA antibody response was not observed in mice immunized with OVA simply mixed with empty FLs. These results indicate that FLs function as a novel immunoadjuvant in inducing antigen-specific antibody production.

Tumor necrosis factor alpha-mediated tumor regression by the in vivo transfer of genes into the artery that leads to tumors.

We report that tumor necrosis factor (TNF) alpha induced a strong antitumor immune reaction when it was produced in arteries leading to tumors by gene transfer in vivo. We used a mouse model carrying a sarcoma-180 tumor in the right footpad and injected the fusogenic liposomes encapsulating the human TNF-alpha gene into the right femoral artery. Under this condition, human TNF-alpha was detected only in the artery leading to the tumor and in the tumor. There was a significant regression in tumor growth when the TNF-alpha gene was delivered into the right femoral artery, with 4 of 11 mice completely cured. No regression was observed when the TNF-alpha gene was delivered into the left femoral artery or into the tumor or when the luciferase gene was administered. Tumor regression was inhibited by the injection of anti-TNF-alpha, anti-CD4, or anti-CD8 monoclonal antibody, and CD8+ T cells accumulated in the tumors of TNF-alpha-treated mice. These results suggest that TNF-alpha expressed locally in the arteries leading to tumors efficiently suppresses tumor growth through reinforcement of an antitumor immune reaction. The significance of this phenomenon for cancer gene therapy was discussed.

Cytoplasmic gene expression system enhances the efficiency of cationic liposome-mediated in vivo gene transfer into mouse brain.

Development of methodologies for gene transfer into the central nervous system (CNS) is important for fundamental research as well as clinical studies for gene therapy. Cationic liposomes (CL) are attractive vectors because of their safety and ease of use. However, to date only low rates of success have been reported. We succeeded in obtaining high transfection efficiencies into the newborn mouse brain in vivo by CL and a cytoplasmic gene expression system based on T7 RNA polymerase and T7 RNA polymerase- and the luciferase-gene with the T7 promoter sequence. This system showed an efficiency rate 2 orders of magnitude higher than the standard system, which used CL and luciferase genes with a Rous sarcoma virus promoter, pRSVL. In addition, in vitro experiments using LLCMK2 cells showed that cytoplasmic gene expression occurred rapidly (within 6 h) after transfection. In contrast, pRSVL required 24-48 h for induction of luciferase expression. Our results suggest that the cytoplasmic gene expression system is useful for gene delivery into the CNS.

Efficient gene transfer into mammalian cells using fusogenic liposome.

Fusogenic liposome (FL) based on Sendai virus constitutes a unique system that delivers the content efficiently into animal cells in vitro and in vivo. In this study we characterized unilamellar FL as a gene transfer vector in comparison with cationic lipid (CL)-DNA complex. FL transferred genes efficiently into cultured cells even when incubated for as little as 10 min, while CL-DNA complex required at least 30 min to reach the same level of gene expression. FL was also much less cytotoxic than CL-DNA complex under the conditions that resulted in the same level of gene expression. In addition, FL maintained 70% of the transfection activity even in the presence of 40% fetal calf serum (FCS), while CL-DNA complex almost completely lost their activity in the presence of 5% FCS. Furthermore, we found that FL could introduce and express luciferase gene into mouse ascites tumor cells in vivo, but CL-DNA complex could not even at higher concentrations of DNA. We conclude that unilamellar FL is a unique and efficient nonviral vector for gene transfer in vitro and in vivo.

[4,4'-(Z)-dehydrophenylalanine]gramicidin S with stabilized bioactive conformation and strong antimicrobial activity.

Dehydrophenylalanine (delta Phe) was incorporated into an antibiotic peptide gramicidin S (GS) in place of D-Phe4,4' to prepare an unsaturated analog. Conformational analysis with 1H-NMR indicated that the unsaturated analog has much the same backbone conformation as that of natural gramicidin S as shown by NOE experiments. Studies on temperature dependences and on the chemical shift differences showed that the hydrogen bonds between Val-NH and Leu-CO in the unsaturated analog are strengthened by the incorporation of delta Phe4,4'. This resulted in the reinforcement of the beta-sheet structure which is the most important structural element for GS bioactivity. [delta Phe4,4']gramicidin S exhibited indeed very strong antimicrobial activities against Gram-positive bacteria as well as the natural peptide.