Abstract 3854: Successful selective eradication of colorectal cancer cells by adenovirus-based delivery of toxins
Authors: Shapira Shiran , Shapira Assaf , Kazanov Dina , Nabiochtchikov Ilana , Kraus Sarah R. , Arber Nadir 
Published: July 2016

Background: K-Ras gene mutation is an early event in the development of colorectal cancer (CRC) and occurs in ∼50% of CRC cases. We propose a strategy that exploits the Ras hyperactive pathway, rather than inhibiting it as was tried and failed many times. We have previously reported that recombinant adenovirus, carrying a pro-apoptotic gene under the regulation of Ras-responsive elements (Ets/AP1), suppressed the growth of cancer cells displaying hyperactive K-Ras (Biomed Pharm, 2005,Cancer Gene Ther,2012, Exp Cell Res,2012). TA systems are evolutionarily successful entities that are prevalent in lower organisms and play important roles in a diverse range of cellular activities.

im: To establish a tight control 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” ΔE1/ΔE3 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-ΔPY4-CMV-MazEF viruses (1×109pfu) 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. The cytotoxic effect was confirmed qualitatively by colony formation assay. 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-MazEF-encoding adenovirus (61%) without any toxic or side effects. Ad-ΔPY4-SV40-MazEF treated mice (control group) tumor volume was reduced only by 27% (P&lt;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.

Citation Format: Shiran Shapira, Assaf Shapira, Dina Kazanov, Ilana Nabiochtchikov, Sarah R. Kraus, Nadir Arber. Successful selective eradication of colorectal cancer cells by adenovirus-based delivery of toxins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3854.