No abstract

Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vector system consisting of two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2 S -M2 and the other has a rtTA responsive promoter driving the expression of b-galactosidase gene (lacZ). The rtTA2 S -M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.

Background and methods: Gene therapy may offer a new tool for the treatment of renal cell carcinoma ( RCC ). We have tested a combination of cytotoxic and antiangiogenic gene therapy for wild -type orthotopic human RCC xenografts in nude mice using intratumoral adenovirus -mediated herpes simplex virus thymidine kinase ( HSV -tk ) and endostatin ( ES ) gene therapy. In vivo magnetic resonance imaging, morphometry, immunocytochemistry, and survival were used to evaluate the treatment effect. Adenovirus -mediated marker gene transfers ( GFP ) were used as controls. Results: In vivo transduction efficiency, measured using GFP gene transfer, was 27 ± 7%. The combination gene therapy with HSV -tk and ES adenoviruses resulted in a significant antitumor effect ( P < .01 ) compared to single HSV -tk ( n.s. ) or ES ( n.s. ). In the survival study, all tumors with single gene therapy using HSV -tk, ES, and marker gene adenoviruses showed progression in magnetic resonance imaging. In contrast, the majority of the tumors in the combination treatment group remained dormant or were eradicated ( 57% ). Survival of these mice equaled healthy nude mice, and was significantly prolonged ( P < .0001 ) compared to HSV -tk ( P < .028 ) and ES ( n.s. ) groups. Conclusions: It is concluded that the inhibition of angiogenesis using ES gene transfer together with the cytotoxic HSV -tk gene therapy results in a significantly improved treatment effect in RCC compared to the single gene treatments.