Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.
Hum Gene Ther. 2021 Feb;32(3-4):216-230. doi: 10.1089/hum.2020.228. Epub 2021 Jan 22.
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. , 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. , 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.
柯萨奇病毒 B3（CVB3）在结直肠癌中有很强的溶瘤活性，但它也会感染胰腺和心脏。为了提高病毒的安全性，我们研究了将靶位（TS）插入病毒基因组是否可以预防胰腺和心脏毒性，这些 TS 与 miR-375 和 miR-1 互补。虽然 miR-375 和 miR-1 在胰腺和心脏中大量表达，但在结直肠癌中它们的表达水平较低，这允许癌细胞被选择性攻击。为了研究 microRNAs 的重要性，我们构建了两种病毒，H3N-375TS 只含有 miR-375TS，而 H3N-375/1TS 含有 miR-375TS 和 miR-1TS。两种病毒均在结直肠癌细胞中复制并裂解，类似于非靶向对照病毒 H3N-39TS，而在瞬时或内源性表达相应 microRNAs 的细胞系中，它们的复制能力显著减弱。此外，对照病毒 H3N-39TS 强烈感染胰腺和心脏，导致在移植了结直肠 DLD-1 细胞肿瘤的小鼠中单次肿瘤内病毒注射后 4 天内发生致命疾病。相比之下，三次肿瘤内注射 H3N-375TS 或 H3N-375/1TS 未能引起病毒引起的疾病。在动物中，两种病毒均从胰腺中完全消除，H3N-375/1TS 也从心脏中消除，而 H3N-375TS 的心脏滴度则大大降低。对 DLD-1 肿瘤模型的长期研究证实，H3N-375TS 和 H3N-375/1TS 处理的小鼠中没有病毒引起的不良反应。没有死亡，胰腺和心脏没有病理改变。关于治疗效果，治疗动物在肿瘤中具有高且持久的 H3N-375TS 和 H3N-375/1TS 持久性，并且肿瘤生长明显减缓。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 毒性的一种非常有效的策略。
