Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region

The use of genetically engineered, replication-selective viruses to treat c ancer is being realized with viruses such as ONYX-015, a human adenovirus t hat selectively destroys p53 mutant cancer cells. To enhance further the cl inical efficacy of ONYX-015 and viruses like it, we have developed a novel gene delivery system for replicating adenoviruses. This system has two uniq ue features. First, it uses the endogenous adenoviral gene expression machi nery (promoter, splicing, polyadenylation) to drive transgene expression. S econd, a single region or gene in the multigene E3 transcription unit is se lectively substituted for by the therapeutic transgene(s). Analyzing variou s transgene substitutions for the 6.7 K/gp19 K region of E3, we demonstrate the following: (1) transgene expression in this system is predictable and mimics the substituted endogenous gene expression pattern, (2) expression o f surrounding E3 genes can be retained, (3) the insertion site choice can e ffect both the transgene expression level and the viral life cycle, and, (4 ) expression levels from this system are superior to those generated from a replication-defective virus using the HCMV enhancer-promoter and this is d ependent on viral DNA replication. This unique methodology has broad applic ation to the rapidly evolving field of replicating virus-based therapies.