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.

Lentiviral vectors encoding rabbit low-density lipoprotein receptor (LDLR) or green fluorescent protein (GFP) under the control of a liver-specific promoter (LSP) were used for intraportal gene transfer into the liver of hypercholesterolemic LDLR-deficient Watanabe Heritable Hyperlipidemic rabbits. In vitro cell culture analysis demonstrated functionality of the LSP-LDLR vector in mediating increased degradation of LDL in transduced liver cells. Twenty-five rabbits were each injected with 1 x 10(9) infectious virus particles into the portal vein. Liver biopsy samples were collected 4 weeks after the gene transfer and the rabbits were followed up for 2 years. Histological and RT-PCR analyses showed the expression of GFP and LDLR transgenes in the biopsy samples. Clinical chemistry and histological analyses revealed normal liver function and morphology during the 2-year follow-up with no safety issues. LSP-LDLR-treated rabbits demonstrated an average of 14 +/- 7% decrease in serum cholesterol levels during the first 4 weeks, 44 +/- 8% decrease at 1 year, and 34 +/- 10% decrease at the 2-year time point compared to the control rabbits. This study demonstrates the safety and potential benefits of the third-generation liver-specific lentiviral vectors in the treatment of familial hypercholesterolemia using direct intraportal liver gene therapy without the need for liver resection.