Evaluation of an E1E4-deleted adenovirus expressing the herpes simplex thymidine kinase suicide gene in cancer gene therapy

Studies with first-generation adenoviral vectors have uncovered limitations that include finite transgene persistence, potential hepatotoxicity, and c ontamination with replication-competent adenovirus (RCA). To address these limitations within the context of cancer suicide gene therapy, a new adenov iral vector was developed containing the herpes simplex virus type 1 thymid ine kinase (HSV tk) gene inserted in the Fl region of a recombinant vector containing deletions in the E-1 and E-4 regions of the Ads genome. The HSV tk minigene was placed under transcriptional control of a Rous sarcoma viru s (RSV) promoter. This new E1E4-deleted vector was compared with the first- generation E1E3-deleted Ad.RSVtk vector. Generation of replication-competen t adenovirus during production was eliminated. Using semiquantitative immun oblotting, the two vectors produced equivalent amounts of the expected 44-k Da tk-encoded protein in three different cell lines tested. The ability of the E1E4-deleted vector to sensitize tumor cells to ganciclovir (GCV) using in vitro assays and mixing studies was comparable to that of the E1E3-dele ted vector. In vivo bystander effects were investigated using mixing studie s in a syngeneic flank tumor model and demonstrated no difference between v ectors in either immunocompetent or immunodeficient mice. To test the effic iency of these vectors in treating tumors in clinically relevant models, vi rus was injected intraperitoneally into tumor-bearing SCID mice and intrapl eurally in a syngeneic rat mesothelioma model. After treatment of animals w ith ganciclovir, both vectors were roughly equivalent in their ability to i ncrease mean survival (from similar to 40 to similar to 70 days) and marked ly reduce tumor burden. Finally, formal toxicology studies were performed a nd showed similar amounts of local inflammation without systemic toxicity. In summary, this series of in vitro and in vivo experiments indicates that the performance of the recombinant E1E4-deleted adenoviral vector was virtu ally identical to that of the E1E3-deleted vector. Since the E1E4 vector ha s a much lower rate of recombination during production and has been shown t o be less hepatotoxic in animal models, this new vector should prove superi or to the first-generation Ad.HSVtk vectors in clinical cancer gene therapy trials.