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Exogenous control of mammalian gene expression through modulation of RNA self-cleavage


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 sequences1,2,3,4. 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 motifs5 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 technologies6, 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.

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Figure 1: Strategy for controlling gene expression through the modulation of RNA self-cleavage and optimization of Schistosome Sm1 ribozyme self-cleavage activity.
Figure 2: Efficient self-cleavage can occur in different cells, with different vectors and with ribozyme sequences positioned in different locations.
Figure 3: Induction of gene expression in cultured cells through inhibition of ribozyme self-cleavage.
Figure 4: Effective control of gene expression in vivo using ribozyme-based gene regulation system.


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We thank K. Salehi-Ashtlani and J. Szostak for helpful discussions, Y. Tang and R. Weissleder for help with imaging experiments performed during the early course of the work, and M. Chung for her technical assistance. This work was supported by grants from AMGEN and L'Association Francaise contre les Myopathies (AFM). R.C.M. is an AMGEN consultant and equity holder.

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Correspondence to Richard C. Mulligan.

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Funding for this work was provided by AMGEN corporation and L'Association Francaise contre les Myopathies (AFM). R.C.M. holds a non-paying consultant position and AMGEN equity.

Supplementary information

Supplementary Table

Survey of ability of different ‘self-cleaving’ ribozymes to function in mammalian cells. Including a list of references. (PDF 106 kb)

Supplementary Methods (DOC 24 kb)

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Yen, L., Svendsen, J., Lee, JS. et al. Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 431, 471–476 (2004).

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