Development of a novel recombinant adenovirus containing gfp-zeocin fusion expression cassette for conditional replication in p53-deficient human tumor cells.

Two obstacles limiting the efficacy of nearly all cancer gene therapy trails are low gene transduction efficiency and the lack of tumor specificity. Fortunately, a replication-competent, E1B-deficient adenovirus (dl1520) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53. In an attempt to appraise the efficiency and safety of this approach, a novel recombinant adenovirus, r3/Ad, containing a gfp-zeocin expression cassette was constructed in this work. The study in vitro demonstrated that r3/Ad has the ability to replicate in and lyse only the p53-deficient human tumor cells such as the human glioblastoma cells (U251) and human bladder cells (EJ) but not in the human fibroblast cells (MRC-5) with functional p53. Importantly, this gfp-zeocin fusion gene driven by the bipromoter (CMV and EM-7) could be used as an effective selective marker and reporter in prokaryotic and eukaryotic cells; and also zeocin as a selective marker could minimize contamination of the recombinant virus by the wt-Ad5. Additionally, it was found that the r3/Ad could be useful for studying the selective replication of E1B-deficient adenovirus in vivo, it could be used as a "guide" to study the ability of the recombinant adenovirus to spread and to infect distant tumor cells in any tumor bearing animal model by GFP as a reporter. This may help determine the safety of using any E1B-deficient adenovirus in cancer gene therapy.

Conditional replication of a recombinant adenovirus studied using neomycin as a selective marker.

An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.