Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

INTRODUCTION: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. MATERIALS AND METHODS: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. RESULTS: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. DISCUSSION: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases.

Effects of mechanical stress on cytokine production in mandible-derived osteoblasts.

OBJECTIVE: Mechanical stress is known to be an important factor in the regulation of bone remodeling, and mandibular bone is continuously exposed to mechanical stressors such as occlusal force. Therefore, in this study, we investigated the effects of mechanical stress approaching occlusal force, to which mandible-derived osteoblasts (MDOB) are exposed, on cytokine expression and production using an original hydrostatic pressure apparatus. MATERIALS AND METHODS: The levels of cytokine in MDOB were examined by real-time RT-PCR, ELISA, and western blotting. In addition, mitogen-activated protein kinase inhibitor for ERK1/2, JNK, and p-38 pathways was used to identify the signal transduction pathway. RESULTS: Hydrostatic pressure increased the expression of IL-6 and TNF-α mRNA in a magnitude- and time-dependent manner and also enhanced IL-6 and TNF-α protein production. Furthermore, hydrostatic pressure changed the RANKL/OPG ratio in favor of RANKL for both mRNA and protein levels. Specific inhibitor of p-38 pathway but not that of the ERK1/2 and JNK pathways suppressed the up-regulation of RANKL production induced by hydrostatic pressure loading. CONCLUSION: These results suggest that MDOB play a role in cytokine production in response to mechanical stress and that occlusal force may support the maintenance of mandible bone homeostasis by activating bone remodeling through osteoclastogenesis.

Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure.

OBJECTIVE: The aim of this study was to investigate the effect of intermittent hydrostatic pressure (IHP) on chondrogenic differentiation of synovium-derived progenitor cells (SPCs). METHODS: SPCs, bone marrow-derived progenitor cells and skin fibroblasts from rabbits were subjected to IHP ranging from 1.0 to 5.0 MPa. The mRNA expression of proteoglycan core protein (PG), collagen type II and SOX-9 was examined using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The production of SOX-9 protein and glycosaminoglycan (GAG) by SPCs was analyzed by Western blot and the dimethylmethylene blue assay. In addition, mitogen-activated protein (MAP) kinase inhibitors for c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and the p38 pathway were used to identify the signal transduction pathways. RESULTS: Real-time RT-PCR showed that mRNA expression of PG, collagen type II and SOX-9 was significantly enhanced only in SPCs receiving 5.0 MPa of IHP. The production of SOX-9 protein and GAG by SPCs was also increased by exposure to 5.0 MPa of IHP. These up-regulated expressions were suppressed by pretreatment with an inhibitor of JNK, but not with inhibitors of ERK or p38. CONCLUSION: Our results demonstrated that the exposure of SPCs to 5.0 MPa of IHP could facilitate induction of the chondrogenic phenotype by the MAP kinase/JNK pathway. This finding suggests the potential for IHP utilization in regenerative treatments for cartilage injuries or osteoarthritis.

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf.

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

Cytokine genetic adjuvant facilitates prophylactic intravascular DNA vaccine against acute and latent herpes simplex virus infection in mice.

Intravascular plasmid DNA (pDNA) vaccine encoding herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) effectively induces prophylactic immunity against lethal HSV-1 infection in mice. We investigated whether the vaccine potency is further improved by coadministration of cytokine genes together with a low dose of genetic vaccine. pDNA encoding IL-12, IL-15, IL-18 or IL-21 was capable of elevating survival rates of HSV-1-infected mice when coinjected with 1 microg of gB pDNA, while IL-10 gene delivery failed to affect the effectiveness of the genetic immunization. Although only 17% of mice survived acute HSV infection after the gB pDNA vaccination at a dose of 1 microg, all mice coadministered with 1 microg each of gB and IL-12 pDNAs not only survived the acute infection but also escaped latent infection. In these animals, the neutralizing antibody against HSV-1 was abundantly produced, and CTL activity against the gB antigen was augmented. Coadministration of the gB and IL-12 genes also elevated the serum level of interferon-gamma. Adaptive transfer experiments indicated that soluble factors contributed to preventive immunity, while cell components alone were not capable of protecting mice from fatal viral infection. These results strongly suggest potential usefulness of Th1 cytokine genes as effective molecular adjuvants that facilitate specific humoral as well as cellular immune responses elicited by intravascular molecular vaccination.

Intravascular naked DNA vaccine encoding glycoprotein B induces protective humoral and cellular immunity against herpes simplex virus type 1 infection in mice.

Naked plasmid DNA (pDNA) vaccine expressing herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) was tested for protective activity against acute HSV-1 infection in mice. The pDNA was intravenously injected into Balb/c mice via their tail vein under high pressure, and the vaccination was performed two times at an interval of 7 days. The gB gene vaccination significantly protected the mice from subsequent intraperitoneal challenge with a lethal dose of HSV-1, which killed all the animals given control plasmid or saline. The protective activity was correlated with the dose of the plasmid inoculated, the survival rate reaching 83% in mice vaccinated with 5 microg of pDNA. The vaccinated mice were also protected from latent HSV infection. The immunized mice showed significant elevation in neutralizing antibody against HSV-1 as well as serum levels of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma). When mice were immunized with 5 microg of an Epstein-Barr virus (EBV)-based plasmid vector harboring the gB, the cytotoxic T lymphocytes (CTLs) activity and proliferative response for HSV-1 were also induced. The results strongly suggest that intravenous immunization of naked pDNA may induce humoral and cellular immune responses against the virus, leading to a significant prophylactic outcome against HSV-1 infection in mice.

Prolonged gene expression in mouse lung endothelial cells following transfection with Epstein-Barr virus-based episomal plasmid.

The development of a strategy to deliver a gene to pulmonary endothelium will be useful for gene function study and for pulmonary gene therapy. Cationic lipidic vectors are efficient in gene transfer to pulmonary endothelium via the vascular route; however, gene expression is transient and lasts for only a few days. In this study, we show that pulmonary gene transfer via cationic lipidic vectors can be significantly improved using an Epstein-Barr virus (EBV)-based expression plasmid. Systemic administration of cationic liposomes followed by the EBV-based plasmid led to gene expression in the lung that lasted for more than 3 weeks. Prolonged and high levels of gene expression can also be obtained in primary mouse lung endothelial cells (MLEC) following lipofection with an EBV-based plasmid. These results suggest the utility of this gene transfer protocol in studying the expression of cloned genes in lung endothelial cells and in pulmonary gene therapy.

Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo.

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.

Improvement of nonviral gene therapy by Epstein-Barr virus (EBV)-based plasmid vectors.

The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the beta-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.

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

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.

In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice.

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.

Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells.

The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Oiagen), linear polyethylenimine (PEI; ExGen 500; Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.beta) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl beta-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal; Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2microg of pSES.beta was combined with either 21 microg of dendrimer, 1.7microg of linear PEI, or 2.0microg of branched PEI. At the same DNA/polymer ratios, the proportions of X-gal-stained cells were also the highest (31.3 +/- 7.5%, 30.3 +/- 9.0%, and 8.3 +/- 3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.

Expression of transduced HSP70 gene protects chondrocytes from stress.

OBJECTIVE: To investigate the efficacy of adenovirus vector mediated transduction of heat shock protein 70 (HSP70) gene to human chondrocyte-like cell (HCS-2/8) against heat stress. METHODS: Two adenovirus vectors that contain wild-type (AxSHEwt) or mutant-type (AxSHEmt) HSP70 gene, and that are regulated by SRalpha promoter, were constructed. The mutant-type lacks the area that expresses stress durability. One of the 2 adenovirus vectors was added to the cultures of human chondrocyte-like cells (HCS-2/8). Heat stress (48 degrees C) was applied to the transduced cells for 2 h, and the efficacy of adenovirus vector mediated transduction of HSP70 gene against heat stress in the chondrocytes was investigated using alamar blue assay and MTT assay. RESULTS: Absorbance levels at 48 degrees C were 300.3 +/- 51.9 and 1.173 +/- 0.011 in the controls, 278.5 + 33.8 and 1.217 +/- 0.018 in the AxSHEmt transduced cells, and 349 +/- 14.7 and 1.371 +/- 0.033 in the AxSHEwt transduced cells. The level in the AxSHEwt transduced cells was significantly higher than in the other 2 groups (p < 0.05). With 37 degrees C treatment, no significant difference was observed. CONCLUSION: Chondrocytes to which HSP70 gene was transduced had a significantly higher metabolic activity and viability under heat stress.

In vivo gene gun-mediated transduction into rat heart with Epstein-Barr virus-based episomal vectors.

BACKGROUND: Gene guns have been used to transfer genes into various organs, but there has been no report of successful gene gun-mediated gene transfer into the heart. In this study, we assessed the possibility of gene therapy using a gene gun and an episomal plasmid vector. METHODS: Gene transfer was performed using two sizes of gold particles and two plasmids (an episomal vector and a conventional plasmid vector). From the first to eighth week after the bombardment, rats were sacrificed. The excised hearts were subjected to X-gal staining and histologic examination. To ensure that plasmid was not distributed to organs other than the heart, the presence of the beta-gal sequence was examined by polymerase chain reaction analyses. RESULTS: Gene expression persisted for 6 weeks. The episomal vector apparently contributed to long-lasting expression. Infiltration of monocytes or leukocytes was very faint. The beta-gal DNA was detected in bombarded hearts but not other organs. CONCLUSIONS: Gene gun-mediated transfer of the episomal vector into beating heart may provide a simple, efficient, and useful strategy for gene therapy.

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.

The present study reports a novel nonviral method to efficiently and specifically target carcinoembryonic antigen (CEA)-producing cholangiocarcinoma (CC) cells in vitro. Epstein-Barr virus (EBV)-based and conventional plasmid vectors were constructed that possess the beta-galactosidase (beta-gal) or herpes simplex virus-1 (HSV-1) thymidine kinase (Tk) genes as well as tandem repeats of the human genomic sequence -82 to -42 bp from the transcriptional start site of the CEA gene. The plasmids were transfected by means of polyamidoamine dendrimer into CEA-positive (HuCC-T1) or -negative cell lines. Transfection of the conventional plasmid vector with the CEA promoter and beta-gal gene resulted in a very low or undetectable level of marker gene expression even in the CEA-positive cell line. Transferring the HSV-1 Tk gene by conventional plasmid did not affect the susceptibility of HuCC-T1 cells to ganciclovir. In marked contrast, strong beta-gal expression was specifically obtained in HuCC-T1 cells by transfecting the EBV-based plasmid in which the CEA promoter and a ubiquitous promoter (SRalpha) are employed to drive the EBV-encoded nuclear antigen 1 (EBNA1) and beta-gal genes, respectively (pTES.beta). Furthermore, CEA-positive but not -negative tumor cells were rendered highly susceptible to ganciclovir when transfected with the EBV-based vector that carries the CEA promoter-EBNA1 and SRalpha-HSV-1 Tk genes (pTES.Tk). These results strongly suggest that the EBV-based plasmid vector/cationic polymer system (EBV/polyplex) equipped with the CEA promoter provides an efficient nonviral method for the targeted gene therapy of CEA-producing malignancies.

Gene delivery to human chondrocytes by an adeno associated virus vector.

OBJECTIVE: To investigate the efficiency of gene transduction to human chondrocytes using an adeno associated virus (AAV) vector. METHODS: We transduced green fluorescent protein (GFP) gene using AAV vector to primary human chondrocytes as well as human cartilage organ cultures, in which chondrocytes are surrounded by extracellular matrix. Expression of GFP gene was analyzed at various time points after transduction by fluorescence microscopy and immunohistochemistry. RESULTS: In primary chondrocytes, the percentages of GFP positive cells were 15.9% or 16.0% on Day 1 and 95.0% or 93.7% on Day 7 after gene transduction. In cartilage organ cultures, gene delivery was observed in cells located not only in the superficial layer but also in the deep layer within the cartilage tissue. Up to 45.3+/-7.4% or 46.0+/-3.9% of chondrocytes expressed GFP for at least 28 days. CONCLUSION: AAV vector could be useful for direct gene delivery to chondrocytes in situ.

Ex vivo gene delivery using an adenovirus vector in treatment for cartilage defects.

OBJECTIVE: To realize local selective gene expression in grafted chondrocytes for cartilage defect, we investigated the usefulness of an ex vivo gene delivery method using an adenovirus vector. METHODS: Beta-galactosidase gene (LacZ) was transfected using an adenovirus vector to chondrocytes isolated from rat joints. The cells were then embedded into collagen gel, and LacZ expression in the gel was examined using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining; beta-galactosidase activity was also measured. The collagen gel containing transfected chondrocytes was grafted to the experimental cartilage defects, and the expression of delivered gene was histologically examined after X-gal staining of the tissue containing the grafted area. RESULTS: X-gal positive chondrocytes in the gel accounted for 82% at one week and 55% at 8 weeks after gene delivery. Beta-galactosidase activity decreased with time, but its expression was maintained even at 8 weeks after gene delivery. Chondrocytes used in the allograft maintained their morphology, and the expression of delivered gene continued during the 8 week period. CONCLUSION: In this ex vivo method, delivered gene can be expressed efficiently for a long time; this method would be useful in allografts for cartilage defects.

Highly efficient suicide gene expression in hepatocellular carcinoma cells by epstein-barr virus-based plasmid vectors combined with polyamidoamine dendrimer.

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 beta-galactosidase gene was transferred to HuH7, PLC/PRF/5, or HLE cells, < or =50-fold higher beta-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 microM), 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.

Effective suicide gene therapy in vivo by EBV-based plasmid vector coupled with polyamidoamine dendrimer.

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.