Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established ac ell-based assay in which expression of a luciferase ( luc)reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter ; 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA selfcleavage.

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