Selective eradication of cancer cells by delivery of adenovirus-based toxins

// Shiran Shapira 1 , Assaf Shapira 2 , Diana Kazanov 1 , Gil Hevroni 1 , Sarah Kraus 1 , Nadir Arber 1 1 Laboratory of Molecular Biology, The Integrated Cancer Prevention Center, Tel Aviv Sourasky Medical Center, Affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel 2 Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel-Aviv, Israel Correspondence to: Nadir Arber, email: nadir@tlvmc.gov.il Keywords: MazEF, toxin-antitoxin, KRAS mutation, cancer gene therapy Received: June 09, 2016      Accepted: March 13, 2017      Published: April 07, 2017 ABSTRACT Background and objective: KRAS mutation is an early event in colorectal cancer carcinogenesis. We previously reported that a recombinant adenovirus, carrying a pro-apoptotic gene ( PUMA ) under the regulation of Ets/AP1 (RAS-responsive elements) suppressed the growth of cancer cells harboring hyperactive KRAS. We propose to exploit the hyperactive RAS pathway, rather than to inhibit it as was previously tried and failed repeatedly. We aim to improve efficacy by substituting PUMA with a more potent toxin, the bacterial MazF-MazE toxin-antitoxin system, under a very tight regulation. Results: A massive cell death, in a dose-dependent manner, reaching 73% at MOI 10 was seen in KRAS cells as compared to 22% in WT cells. 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. Considerable tumor shrinkage (61%) was demonstrated in vivo following MazEF-encoding adenovirus treatment without any side effects. Design: Efficient vectors for cancer-directed gene delivery were constructed; “pAdEasy-Py4-SV40mP-mCherry-MazF”“pAdEasy-Py4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP“,“pAdEasy-ΔPy4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP “and “pAdEasy-mCherry”. Virus particles were produced 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 apoptotic and dead cells, respectively. In vivo tumor formation was measured in a xenograft model. Conclusions: A proof of concept for a novel cancer safe and effective gene therapy exploiting an aberrant hyperactive pathway is achievable.