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To accomplish efficient nonviral gene therapy against prostate cancer (PC), Epstein-Barr virus (EBV)-based plasmid vectors containing EBNA1 gene and oriP were employed and combined with a cationic polymer or cationic lipid. When EBV-plasmid/poly-amidoamine dendrimer complex was injected into PC-3-derived tumors established in severe combined immunodeficiency mice, a considerable expression of marker gene was obtained in the tumors, and the expression level was more than eight-fold higher than that achieved by conventional plasmid vector/dendrimer. Since most PC cells express the apoptotic signal molecule Fas (Apo-1/CD95) on their surface, Fas ligand (FasL) gene was transferred into PC cells to kill the tumor cells. In vitro transfection with pGEG.FasL (an EBV-plasmid with the FasL gene) significantly reduced the viability of PC cells, which subsequently underwent apoptosis. Intratumoral injections of pGEG.FasL into PC induced significant growth suppression of the xenograft tumors, in which typical characteristics of apoptosis were demonstrated by TUNEL staining and electron microscopic observations. When pGEG.FasL transfer was accompanied by systemic administrations of cisplatin, the tumors were inhibited even more remarkably, leading to prolonged survival of the animals. FasL gene transfection by means of EBV-based plasmid/cationic macromolecule complexes may provide a practical therapeutic strategy against PC.

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.

Direct intratumoral transfection of cytokine genes was performed by means of the in vivo electroporation as a novel therapeutic strategy for cancer. Plasmid vectors carrying the firefly luciferase, interleukin (IL)-12 and IL-18 genes were injected into established subcutaneous B16-derived melanomas followed by electric pulsation. When plasmid vectors with Epstein--Barr virus (EBV) nuclear antigen 1 (EBNA1) gene were employed, the expression levels of the transgenes were significantly higher in comparison with those obtained with conventional plasmid vectors. In consequence of the transfection with IL-12 and IL-18 genes, serum concentrations of the cytokines were significantly elevated, while interferon (IFN)-gamma also increased in the sera of the animals. The IL-12 gene transfection resulted in significant suppression of tumor growth, while the therapeutic effect was further improved by co-transfection with IL-12 and IL-18 genes. Repetitive co-transfection with IL-12 and IL-18 genes resulted in significant prolongation of survival of the animals. Natural killer (NK) and cytotoxic T lymphocyte (CTL) activities were markedly enhanced in the mice transfected with the cytokine genes. The present data suggest that the cytokine gene transfer can be successfully achieved by in vivo electroporation, leading to both specific and nonspecific antitumoral immune responses and significant therapeutic outcome.

In chronic heart failure, down-regulation of beta-adrenergic receptor (beta-AR) occurs in cardiomyocytes, resulting in low catecholamine response and impaired cardiac function. To correct the irregularity in the beta-AR system, beta-AR gene was transduced in vivo into failing cardiomyocytes. The Epstein-Barr virus (EBV)-based plasmid vector carrying human beta2-AR gene was injected into the left ventricular muscle of Bio14.6 cardiomyopathic hamsters whose beta-AR is down-regulated in the cardiomyocytes. The echocardiographic examinations revealed that stroke volume (SV) and cardiac output (CO) were significantly elevated at 2 to 4 days after the beta2-AR gene transfer. Systemic loading of isoproterenol increased the cardiac parameters more significantly on day 2 to day 7, indicating that the adrenergic response was augmented by the genetic transduction. The same procedure did not affect the cardiac function of normal hamsters. Immunohistochemical examinations demonstrated human beta2-AR expression in failing cardiomyocytes transduced with the gene. RT-PCR analysis detected mRNA for the transgene in the heart but not in the liver, spleen, or kidney. The procedures may provide a feasible strategy for gene therapy of severe heart failure. Gene Therapy (2000) 7, 2087-2093.

This study demonstrates in vivo effectiveness of a nonviral vector system, Epstein-Barr virus (EBV)-based plasmid vector coupled with polyamidoamine (PAMAM) dendrimer (EBV/polyplex), in suicide gene therapy of cancer. The EBV-based vector is a plasmid vector containing EBV nuclear antigen 1 (EBNA1) gene and oriP from EBV genome. HSV-1 tk gene was transferred into Ewing's sarcoma cell lines, A4573 and KP-EWS-YI, by using an EBV-based plasmid vector, pSES.Tk, or a conventional plasmid vector, pS.Tk. Cells transfected with pSES.Tk/dendrimer showed approximately 100 times lower ID50 to ganciclovir (GCV) compared with those transfected with pS. Tk/dendrimer. Intratumoral injection of pSES.Tk/dendrimer but not pS. Tk/dendrimer drastically suppressed the growth of tumors which had generated from A4573 or Huh7 hepatocellular carcinoma (HCC) cells inoculated into severe combined immunodeficiency (SCID) mice. The treatment with pSES.Tk/dendrimer also resulted in significant prolongation of survival of the mice implanted with A4573. These results suggest that the EBV/polyplex system could be useful for in vivo suicide gene therapy of cancer. Gene Therapy (2000) 7, 53-60.