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ACS Synth Biol. 2019 Aug 15. doi: 10.1021/acssynbio.9b00177. [Epub ahead of print]

Reversible Gene Regulation in Mammalian Cells Using Riboswitch-Engineered Vesicular Stomatitis Virus Vector.

Author information

1
Nucleic Acid Chemistry and Engineering Unit , Okinawa Institute of Science and Technology Graduate University , Onna , Okinawa 904 0495 , Japan.

Abstract

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.

KEYWORDS:

RNA replicon; RNA virus; aptazyme; riboswitch

PMID:
31415142
DOI:
10.1021/acssynbio.9b00177

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