Highly efficient suicide gene expression in hepatocellular carcinoma cells by Epstein-Barr virus-based plasmid vectors combined with polyamidoamine dendrimer

Abstract

The present study was aimed at devising an efficient nonviral strategy for suicide gene therapy of hepatocellular carcinoma (HCC). To improve the efficiency of DNA delivery and expression, we applied Epstein-Barr virus (EBV)-based plasmid vectors instead of conventional plasmid vectors and combined them with cationic liposome (EBV/lipoplex) or polyamidoamine dendrimer (PAAD) (EBV/polyplex). When the ␤-galactosidase gene was transferred to HuH7, PLC/PRF/5, or HLE cells, Յ50-fold higher ␤-galactosidase activities were demonstrated in the cells transfected with EBV vector compared with those transfected with conventional plasmid vectors. PAAD-mediated transfection of HCC with pSES.Tk (an EBV-based vector carrying the herpes simplex virus-1 thymidine kinase gene) resulted in a marked reduction in viable cell number by the addition of ganciclovir (GCV). The HCC cells transfected with pSES.Tk/PAAD showed 100-to 1000-fold higher susceptibilities to GCV than those transfected with pS.Tk (a conventional plasmid vector carrying herpes simplex virus-1 thymidine kinase gene)/PAAD. The pSES.Tk-transfected HCC cells were effectively killed by day 9 in culture with a clinically feasible concentration of GCV (25 M), whereas the pS.Tk-transfected cells survived the culture. These results demonstrate highly efficient suicide gene transfer into various HCC cells by EBV-based plasmid vectors in vitro, suggesting the possible application of this nonviral vector system to gene therapy of HCC. Cancer Gene Therapy (2000) 7, 27-36

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…By 9 h after calcium was restored, cultures pretreated and infected in calcium-free DMEM supplemented with 100 mM EGTA accumulated fluorescein as well as control cultures (Figure 6b; 9 h), indicating that paracellular junction integrity was restored by this time point. No further change in the pattern of fluorescein accumulation was observed by 24 ( Figure 6b; 24 h), 48 , and 72 h after calcium restoration (data not shown). No differences in fluorescein accumulation were observed whether hepatocyte cultures were mock-infected or infected with baculovirus (data not shown).…”

“…Many different gene delivery methods are available including cationic lipid-based DNA transfection [44][45][46][47][48][49] and retroviral and adenoviral gene transfer vectors; [50][51][52][53][54][55] however, the use of these methods for hepatocytes has not been effective because hepatocytes do not divide, are highly susceptible to toxicity, and transfection efficiencies in hepatocytes are extremely low (HC Isom, unpublished data). 11 Previous studies have reported baculovirus-mediated gene delivery to primary hepatocytes.…”

Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by polyamidoamine dendrimer-mediated transfer of an Epstein-Barr virus (EBV)-based plasmid vector carrying the CEA promoter

“…Recently, we have reported that use of the EBV-based plasmid vector combined with polyamidoamine dendrimer (PAAD) (EBV/polyplex) resulted in a markedly high expression in human HCC and Ewing's sarcoma cell lines, suggesting the potential usefulness of the EBV/polyplex system for gene therapy. 31,32 However, no study has been reported in which a tumor-specific promoter was employed in the EBVbased plasmid vector to target tumor cells. In this study, we combined the multimerized CEA promoter with the EBV/polyplex system to establish an efficient nonviral strategy to target CEA-producing cancers.…”

“…Four plasmid vectors, pSES.␤, pS.␤, pSES.Tk, and pS.Tk (Figs 2 and 3), have been described previously. 21,31 The other plasmid vectors, pT.␤, pTES.␤, pSET.␤, pTET.␤, pT.Tk, and pTES.Tk (Figs 2 and 3), were constructed by substituting the tetraplicated CEA promoter for the SR␣ promoter in pS.␤, pSES.␤, pS.Tk, or pSES.Tk.…”

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein–Barr virus (EBV)-based plasmid vectors

“…We have applied the EBV-plasmids to various preclinical studies of gene therapy, including those for hereditary, 21 chronic, 22,23 as well as malignant diseases. [24][25][26][27] In some of these studies, the EBV-based plasmid vectors were combined with cationic liposomes, 25,28 cationic polymers, 23,[25][26][27]29 electroporation 21 or gene gun, 30 while in the other studies, naked EBV-based plasmid was injected into the cardiac muscle. 22,29 In the present study, we transferred the naked EBV-based plasmid vectors to the liver through the intravascular route.…”