Reversible Gene Regulation in Mammalian Cells Using Riboswitch-Engineered Vesicular Stomatitis Virus Vector
Reversible Gene Regulation in Mammalian Cells Using Riboswitch-Engineered Vesicular Stomatitis Virus Vector
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DOI：
10.1021/acssynbio.9b00177
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发表时间：
2019-09-01
影响因子：
4.7
通讯作者：
Yokobayashi, Yohei
中科院分区：
文献类型：
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作者：
Takahashi, Kei;Yokobayashi, Yohei
基于嵌入非翻译区 (UTR) 的小分子响应性自裂解核酶（适体酶）的合成核糖开关可以对哺乳动物细胞中的基因表达进行化学控制。在这项工作中，我们使用鸟嘌呤响应适体酶来控制复制无能的水泡性口炎病毒（VSV）载体的转基因表达。 VSV 是一种无节段、负义、胞质 RNA 病毒，无需 DNA 中间体即可复制，其在疫苗和溶瘤病毒治疗中的应用正在探索中。通过在病毒基因和转基因的 3' UTR 中插入鸟嘌呤激活的核酶，在鸟嘌呤存在的情况下，哺乳动物细胞中 VSV 载体的 GFP 表达被抑制高达 26.8 倍。此外，我们通过在 12 天内从培养基中添加和去除鸟嘌呤，证明了转基因（分泌的 NanoLuc）的可逆调节。总之，我们的核糖开关控制的 VSV 载体可以对哺乳动物细胞中的基因表达进行稳健、长期、长期和可逆的调节，而不会产生不良基因组整合的风险。
Synthetic riboswitches based on small molecule responsive self-cleaving ribozymes (aptazymes) embedded in the untranslated regions (UTRs) allow chemical control of gene expression in mammalian cells. In this work, we used a guanine-responsive aptazyme to control transgene expression from a replication incompetent vesicular stomatitis virus (VSV) vector. VSV is a nonsegmented, negative-sense, cytoplasmic RNA virus that replicates without DNA intermediates, and its applications for vaccines and oncolytic viral therapy are being explored. By inserting the guanine activated ribozyme in the 3' UTRs of viral genes and transgenes, GFP expression from the VSV vector in mammalian cells was repressed by as much as 26.8-fold in the presence of guanine. Furthermore, we demonstrated reversible regulation of a transgene (secreted NanoLuc) by adding and withdrawing guanine from the medium over the course of 12 days. In summary, our riboswitch-controlled VSV vector allows robust, long-term, long-term, and reversible regulation of gene expression in mammalian cells without the risk of undesirable genomic integration.