Takahashi Kei, Yokobayashi Yohei
Nucleic Acid Chemistry and Engineering Unit , Okinawa Institute of Science and Technology Graduate University , Onna , Okinawa 904 0495 , Japan.
ACS Synth Biol. 2019 Sep 20;8(9):1976-1982. doi: 10.1021/acssynbio.9b00177. Epub 2019 Aug 15.
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, and reversible regulation of gene expression in mammalian cells without the risk of undesirable genomic integration.
基于嵌入非翻译区（UTR）的小分子响应性自我切割核酶（适体酶）的合成核糖开关能够对哺乳动物细胞中的基因表达进行化学调控。在这项研究中，我们使用了一种鸟嘌呤响应性适体酶来控制来自无复制能力的水疱性口炎病毒（VSV）载体的转基因表达。VSV是一种不分节段的、负链、细胞质RNA病毒，其复制过程无需DNA中间体，目前正在探索其在疫苗和溶瘤病毒治疗方面的应用。通过将鸟嘌呤激活的核酶插入病毒基因和转基因的3'UTR中，在存在鸟嘌呤的情况下，哺乳动物细胞中VSV载体的绿色荧光蛋白（GFP）表达被抑制了多达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，证明了对转基因（分泌型纳米荧光素酶）的可逆调控。总之，我们的核糖开关控制的VSV载体能够在哺乳动物细胞中对基因表达进行强大、长期且可逆的调控，而不存在不良基因组整合的风险。
