Coxsackievirus B3 (CVB3) has strong oncolytic activity in colorectal carcinoma but it also infects the pancreas and the heart. To improve the safety of the virus, here we investigated whether pancreas and cardiac toxicity can be prevented by insertion of target sites (TS), which are complementary to miR-375 and miR-1 into the viral genome. Although miR-375 and miR-1 are abundantly expressed in the pancreas and in the heart, respectively, their expression levels are low in colorectal carcinomas, which allows the carcinomas to be selectively attacked. To investigate the importance of the microRNAs, two viruses were engineered, H3N-375TS containing only miR-375TS and H3N-375/1TS containing miR-375TS and miR-1TS. In vitro, both viruses replicated in and lysed colorectal carcinoma cells, similar to a nontargeted control virus H3N-39TS, whereas they were strongly attenuated in cell lines transiently or endogenously expressing the corresponding microRNAs. In vivo, the control virus H3N-39TS induced strong infection of the pancreas and the heart, which led to fatal disease within 4 days after a single intratumoral virus injection in mice xenografted with colorectal DLD-1 cell tumors. In contrast, three intratumoral injections of H3N-375TS or H3N-375/1TS failed to induce virus-induced sickness. In the animals, both viruses were completely ablated from the pancreas and H3N-375/1TS was also ablated from the heart, whereas the cardiac titers of H3N-375TS were strongly reduced. Long-term investigations of the DLD-1 tumor model confirmed lack of virus-induced adverse effects in H3N-375TS- and H3N-375/1TS-treated mice. There was no mortality, and the pancreas and the heart were free of pathological alterations. Regarding the therapeutic efficiency, the treated animals showed high and long-lasting H3N-375TS and H3N-375/1TS persistence in the tumor and significantly slower tumor growth. These data demonstrate that miR-375- and miR-1-mediated virus detargeting from the pancreas and heart is a highly effective strategy to prevent toxicity of oncolytic CVB3.

中文翻译：

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miR-375 和 miR-1 调节的柯萨奇病毒 B3 对胰腺和心脏没有毒性，但在结直肠癌中具有很强的抗肿瘤效率

柯萨奇病毒 B3 (CVB3) 在结直肠癌中具有很强的溶瘤活性，但它也感染胰腺和心脏。为了提高病毒的安全性，我们在此研究了是否可以通过将与 miR-375 和 miR-1 互补的靶位点 (TS) 插入病毒基因组来预防胰腺和心脏毒性。尽管 miR-375 和 miR-1 分别在胰腺和心脏中大量表达，但它们在结直肠癌中的表达水平较低，这使得癌症可以被选择性地攻击。为了研究 microRNA 的重要性，设计了两种病毒，即仅含有 miR-375TS 的 H3N-375TS 和含有 miR-375TS 和 miR-1TS 的 H3N-375/1TS。体外，这两种病毒在结肠直肠癌细胞中复制并裂解，类似于非靶向对照病毒 H3N-39TS，而它们在瞬时或内源性表达相应 microRNA 的细胞系中被强烈减毒。体内, 对照病毒 H3N-39TS 引起胰腺和心脏的强烈感染，这导致在异种移植结直肠 DLD-1 细胞肿瘤的小鼠中单次瘤内病毒注射后 4 天内导致致命疾病。相比之下，H3N-375TS 或 H3N-375/1TS 的三个肿瘤内注射未能诱导病毒引起的疾病。在动物中，两种病毒都从胰腺中完全消融，H3N-375/1TS 也从心脏中消融，而 H3N-375TS 的心脏滴度显着降低。对 DLD-1 肿瘤模型的长期研究证实，在 H3N-375TS 和 H3N-375/1TS 治疗的小鼠中没有病毒诱导的副作用。没有死亡，胰腺和心脏没有病理改变。关于治疗效率，接受治疗的动物在肿瘤中表现出高而持久的 H3N-375TS 和 H3N-375/1TS 持久性，并且肿瘤生长显着减慢。这些数据表明 miR-375 和 miR-1 介导的病毒从胰腺和心脏脱靶是预防溶瘤 CVB3 毒性的高效策略。