Shapira Shiran, Shapira Assaf, Kazanov Diana, Hevroni Gil, Kraus Sarah, Arber Nadir
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.
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel-Aviv, Israel.
Oncotarget. 2017 Jun 13;8(24):38581-38591. doi: 10.18632/oncotarget.16934.
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.
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.
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.
A proof of concept for a novel cancer safe and effective gene therapy exploiting an aberrant hyperactive pathway is achievable.
KRAS突变是结直肠癌致癌过程中的早期事件。我们之前报道过，一种重组腺病毒，携带在Ets/AP1（RAS反应元件）调控下的促凋亡基因（PUMA），可抑制携带高活性KRAS的癌细胞生长。我们提议利用高活性RAS途径，而不是像之前反复尝试但失败那样去抑制它。我们旨在通过在非常严格的调控下用一种更强效的毒素——细菌MazF-MazE毒素-抗毒素系统替代PUMA来提高疗效。
与野生型（WT）细胞中的22%相比，在KRAS细胞中观察到大量细胞死亡，呈剂量依赖性，在感染复数（MOI）为10时达到73%。MazE（抗毒素）表达的增加保护正常细胞免受该系统任何可能的内部或外部泄漏影响，并证实了靶向系统的选择性、特异性和安全性。在体内，编码MazEF的腺病毒治疗后肿瘤显著缩小（61%），且无任何副作用。
构建了用于癌症导向基因递送的高效载体；“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”和“pAdEasy-mCherry”。产生病毒颗粒并测试其效力。通过荧光显微镜和集落形成试验定性测量细胞死亡，并通过MTT定量。分别使用膜联蛋白V和RedDot2染料进行流式细胞术分析以测量凋亡细胞和死亡细胞。在异种移植模型中测量体内肿瘤形成情况。
利用异常高活性途径进行新型癌症安全有效基因治疗的概念验证是可行的。
