Development of a Selective Non-Viral Gene Directed Enzyme/Pro-Drug Cancer Therapy

POLICASTRO, LUCÍA L.; IBAÑEZ, IRENE L.; DURÁN, HEBE A.; PODHAJCER, OSAVALDO L.

Seattle, Washington

Congreso; 10th Annual Meeting of the American Society of Gene Therapy (ASGT); 2007

ASGT

Reactive oxygen species (ROS) are products of normal aerobic cell metabolism. Imbalance between ROS production and removal produces a pro-oxidant state that has been described as a characteristic of human cancer compared to normal tissues. We hypothesized that the persistent oxidative stress of cancer cells can be used as a differential feature of cancer microenvironment to develop a selective therapy. We selected three DNA sequences that were previously described as redox responsive: a -80,-50 GC - rich motif from the VEGF-A promoter (VE), six repeated CArG motives from the Egr-1 promoter (E6) and a -2002, -1546 region from the matrix metalloproteinase-1 promoter (MMP-1). These sequences were cloned upstream of the reporter luciferase gene. Colon cancer (LoVo) and melanoma (A375/6N) cells were transiently transfected with the different constructs and exposed to exogenous H2O2 generated by the glucose/glucose oxidase (G/GO) system. While all constructs showed a significant response to H2O2, VE showed the largest inducible response. Next, we decided to construct a chimeric responsive promoter by combining VE with E6 because the latter can be additionally activated by ionizing radiation. Among different combinations, the hybrid 5´E6(40)VE3´, where 40 is the distance in base pairs between both motives, showed the largest inducible response to exogenous H2O2. In correlation with the 2-5 fold increased ROS levels observed in malignant cells, E6(40)VE-LUC activity was higher in cancer cells than in their respective normal counterparts. Moreover, co-transfection with a plasmid encoding catalase or addition of the exogenous protein reduced the responsiveness of E6(40)VE-LUC to endogenous ROS levels and to increased levels of exogenous H2O2, respectively. This confirmed that E6(40)VE-LUC activity was largely dependent on oxidative stress levels. Next, we cloned the thymidine kinase gene (TK) downstream of 5´E6(40)VE3´ to generate E6(40)VE-TK. Transient expression of TK driven by 5´E6(40)VE3´ in LoVo and A375/6N cells followed by GCV administration led to a significant inhibition of cell growth in vitro, when cells were grown both as monolayers (55-65%) and as spheroids (70-80%). Nude mice xenotransplanted either with LoVo or A375/6N cells transiently transfected with E6(40)VE-TK followed by GCV did not develop tumors at all, or exhibited a significantly reduced tumor growth, respectively. More importantly, E6(40)VE-TK/GCV followed by electroporation was potent enough to exert a therapeutic effect on established LoVo and A375/6N tumors. Moreover, E6(40)VE-TK combination with chemotherapeutic drugs showed enhanced inhibition of tumor cell growth in vitro in cell monolayers and spheroids and in vivo on established tumors compared to either agent alone. Our work demonstrates for the first time that pro-oxidant states of cancer cells and tissues can be used to develop a selective therapeutic strategy, with implications for gene therapy not only in cancer but also in other diseases as well. Publicado en Molecular Therapy, 15(Suppl. 1): 611, 2007