740 Risk of Developing Colorectal Cancer Following a Negative Colonoscopy: A Population-Based Study in Utah

and improved ras responsive element based on the bacterial MazEF system Methods: Efficient vectors for cancer-directed gene delivery were constructed and cloned into a "first generation" DE1/DE3 human type-5 adenoviral-vector.Virus particles were produced, their titer was calculated by the End-Point Dilution Assay (EPDA) and their potency was tested.Cell death was measured qualitatively by using the fluorescent microscopy and colony formation assay, and was quantified by MTT.FACS analysis using annexin V and RedDot2 dyes was performed for measuring apoptosis and dead cells, respectively.In vivo tumor formation was measured in xenograft model.Ad-Py4-SV40-MazEF and Ad-DPY4-CMV-MazEF viruses (1×10 9 pfu) or PBS were administrated intraperitoneal twice with a 3-day interval between injections.Results: Adenovirus therapy induced massive cell death, in a dose-dependent manner; 73% with a titer of 10 MOI in cells with activated K-Ras as compared to 22% in tumor cells having the WT K-Ras (Fig. 1).The cytotoxic effect was confirmed qualitatively by colony formation assay (Fig. 2).In the absence of K-ras-responsive DNA element increase expression of MazE, the anti-toxin, protected normal cells from any possible internal or external leakage of the system and confirmed the selectivity, specificity and safety of the targeting system.FACS analysis confirmed massive cell death, 55% apoptosis and 82% dead cells, following infection with the full toxin-antitoxin encoding viruses.Control viruses lacking the K-ras responsive element a modest toxicity was seen (18% and 10%, respectively).Impressive tumor shrinkage was demonstrated in vivo following treatment with Ad-Py4-SV40-MazEFencoding adenovirus (61%) (Fig. 3) without any toxic or side effects.Ad-DPY4-SV40-MazEF treated mice (control group) tumor volume was reduced only by 27% (P<0.05).No growth inhibition was seen following injection of PBS.Conclusions: A proof-of-concept for a novel cancer gene therapy by exploiting aberrant K-Ras hyperactive pathway was successfully demonstrated.The lack of toxicity holds promise for effective and safe therapy of human cancers carrying K-Ras mutations.