Shusei Ikegami

Adenovirus mediated prostate specific enzyme prodrug gene therapy using prostate specific antigen promoter enhanced by the Cre-loxP system

PURPOSE Tissue or tumor specific gene delivery is crucial for achieving successful results in suicide gene therapy. Prostate specific antigen (PSA) promoter is known to be highly specific in prostate tissue but its promoter activity is much weaker than that of constitutive viral promoters. In the current study we enhanced PSA promoter activity by combining it with the Cre-loxP system. We also applied this system to adenovirus mediated suicide gene therapy with the cytosine deaminase (CD) gene. MATERIALS AND METHODS The Cre-loxP DNA recombination system was used to enhance PSA promoter. A plasmid with the PSA promoter-enhancer combination was constructed to drive Cre recombinase. Another plasmid contained the cytomegalovirus promoter-loxP-flanked stop signal-luciferase gene. LNCaP human prostate cancer cells were co-transfected with these 2 plasmids and luciferase activity was measured to assess promoter activities. Adenoviral vectors with the CD suicide gene were constructed in similar fashion and tested in LNCaP cells in in vitro/in vivo prostate cancer models. RESULTS Promoter activity of the combined PSA promoter/enhancer and Cre-loxP system was 3 times stronger than that of PSA promoter/enhancer alone. It was further enhanced 7-fold in the presence of testosterone. Application of this system to CD suicide gene therapy by adenoviral vectors inhibited subcutaneous LNCaP tumor growth in nude mice. CONCLUSIONS Combining the Cre-loxP system with PSA promoter/enhancer amplified promoter activity and was found to inhibit the growth of PSA producing prostate cancer cells in vivo.

Treatment efficiency of a suicide gene therapy using prostate-specific membrane antigen promoter/ enhancer in a castrated mouse model of prostate cancer

Suicide gene therapy has potential for the treatment of prostate cancer under conditions of androgen deprivation. We show here that the combination of promoter/enhancer of prostate‐specific membrane antigen (PEPM) and the Cre‐loxP system is a good method to express a suicide gene, namely herpes virus thymidine kinase (TK), in prostate cancer cells. We have examined this system in a castration model in vivo, in comparison with a prostate‐specific antigen promoter/enhancer system (PP). In the castrated mice, the tumor luciferase activity with the combination of the PEPM plus the Cre‐loxP system was about 50 times greater than that with the control GL3 plasmid. A similar increase was observed in non‐castrated mice. In contrast, the luciferase activity of the plasmid PP was decreased significantly in tumors from castrated mice as compared with tumors from non‐castrated control mice. Regarding the therapeutic effect, the combination plasmid PEPM‐Cre plus CMV‐loxP‐TK exhibited a strong inhibitory effect on tumor growth in the castrated mice, as in the non‐castrated mice. In contrast, PP‐TK plasmid did not show any significant growth inhibition in the castrated mice. These findings indicate that the combination of PEPM and Cre‐loxP system may have a good treatment effect under androgen ablation conditions in vivo, and our system may therefore be applicable to patients who have previously received androgen deprivation therapy.

Development of Gene Therapy Using Prostate‐specific Membrane Antigen Promoter/Enhancer with Cre Recombinase/LoxP System for Prostate Cancer Cells under Androgen Ablation Condition

To enhance the efficacy of suicide gene therapy for prostate cancer under androgen deprivation, we designed a promoter system that consists of the prostate‐specific membrane antigen (PSMA) promoter/enhancer (PEPM) and Cre‐loxP DNA recombination system. We constructed two kinds of plasmids. One plasmid contains a Cre recombinase (Cre) under the control of PEPM and the other expresses CMV‐lox‐luciferase/herpes simplex virus thymidine kinase (TK). In PSMA‐positive LNCaP cells, the promoter activity of the PEPM‐Cre plus CMV‐lox‐luciferase demonstrated 800‐fold greater activity compared with that of the PSMA promoter alone. However, no enhancement of the promoter activity was observed in the PSMA‐negative cells. Furthermore, in contrast to prostate specific antigen promoter/enhancer (PP), the promoter activity of PEPM did not decrease when the LNCaP cells were cultured in charcoal‐stripped fetal bovine serum (CFBS). In an in vitro gene therapy model with LNCaP cells, the cell growth inhibition in the presence of ganciclovir (GCV) was more evident in the cells transfected with the PEPM‐Cre plus CMV‐lox‐TK than in the cells with the PP‐TK, and the difference in efficacy between the two plasmids was more remarkable when the cells were maintained in CFBS medium. The therapeutic effect of PEPM‐Cre plus CMV‐lox‐TK was also observed in xenografted LNCaP cells on nude mice when the plasmids were directly injected into tumors and GCV was administered intraperitoneally. These findings indicate that the combination of the PSMA promoter/enhancer and the Cre‐loxP system can enhance the PSMA promoter activity even under androgen ablation conditions and can exert its anti‐tumor effect both in vitro and in vivo.

Selective gene therapy for prostate cancer cells using liposomes conjugated with IgM type monoclonal antibody against prostate-specific membrane antigen

Prostate cancer cells express prostate-specific membrane antigen (PSMA). We developed an IgM type monoclonal antibody against PSMA. The antibody was coupled to poly-L-lysine and thereafter this conjugate was mixed with cationic liposomes containing plasmid DNA. The antibody-liposome complex was tested whether it could deliver the gene of interest selectively to the PSMA positive cells. As assessed by β-galactosidase reporter gene, the transfection efficiency was 13.2% with anti-PSMA-liposome complex as compared to 4% with control IgM liposome complex. In contrast., no such differences were observed in PSMA negative PC-3, DU145 and T24 cells. Furthermore, in the suicide gene therapy in vitro with thymidine base gene plus ganciclovir system, anti-PSMA liposome complex demonstrated a selective growth inhibitory effect on PSMA positive LNCaP cells but not on PSMA negative cell lines.

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