Replicating adenoviral vector-mediated transfer of a heat-inducible doublesuicide gene for gene therapy

Tumor cells that express a fusion gene of Escherichia coli cytosine deamina se (CD) and herpes simplex virus type I thymidine kinase (TK) sequences act ivate and are subsequently killed by the nontoxic prodrugs 5-fluorocytosine and ganciclovir. We have previously developed a recombinant adenovirus con taining the CD - TK fusion gene controlled by the human inducible heat shoc k protein 70 promoter so that heat at 41 degreesC for I hour induces therap eutic gene expression. This adenovirus effectively transduces heat-inducibl e expression of the CD-TK gene into human prostate carcinoma cells. However , because a limited number of cells in a tumor can actually be infected, we created a replicating adenoviral vector to increase CD-TK gene expression. This vector is a replication-competent, E1B-attenuated adenoviral vector c ontaining the hsp70 promoter-driven CD-TK gene (Ad.E1A(+)HS-CDTK). When hum an prostate adenocarcinoma DU-145 cells (mutant p53) were infected with the virus at a multiplicity of infection (MOI) of I or 10, the viral replicati on was detected within 2 days at both MOIs. Similar results were observed i n human colorectal carcinoma CX-I cells. When DU-145 cells were infected wi th the virus at an MOI of 10, incubated for 24 hours, heated at 41 degreesC for 4 hours and then harvested 20 hours later, Western blot analysis demon strated that this virus successfully produced viral EIA proteins and heat s hock stimulated the CD-TK gene expression by 12.3-fold. In addition, Ad.E1A (+)HS-CDTK effectively suppressed cell proliferation by viral cytopathic, e ffect). Unlike with a replication-incompetent virus (Ad.HS-CDTK), the cytop athic effect of the virus and cytotoxicity in the presence of the prodrugs were still observed even at low MOI (MOI=1.0).