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Small-molecule-based regulation of RNA-delivered circuits in mammalian cells

Nature Chemical Biology volume 14, pages 1043–1050 (2018)Cite this article

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Abstract

Synthetic mRNA is an attractive vehicle for gene therapies because of its transient nature and improved safety profile over DNA. However, unlike DNA, broadly applicable methods to control expression from mRNA are lacking. Here we describe a platform for small-molecule-based regulation of expression from modified RNA (modRNA) and self-replicating RNA (replicon) delivered to mammalian cells. Specifically, we engineer small-molecule-responsive RNA binding proteins to control expression of proteins from RNA-encoded genetic circuits. Coupled with specific modRNA dosages or engineered elements from a replicon, including a subgenomic promoter library, we demonstrate the capability to externally regulate the timing and level of protein expression. These control mechanisms facilitate the construction of ON, OFF, and two-output switches, with potential therapeutic applications such as inducible cancer immunotherapies. These circuits, along with other synthetic networks that can be developed using these tools, will expand the utility of synthetic mRNA as a therapeutic modality.

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Fig. 1: Small-molecule-based regulation of expression from modRNA.
Fig. 2: Small-molecule-based regulation of expression from replicons.
Fig. 3: Small-molecule-based ON switches driven from replicons.
Fig. 4: Small-molecule-based two-output switches driven from replicons.

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Data availability

The authors declare that data supporting the finding of this study are available within the article and its Supplementary Information. Sample analysis of cytometry data can be found in Supplementary Fig. 29. Replicon MoClo assembly plasmids have been submitted to Addgene with the accession numbers 115928, 115929, 115930, 115931, 115932, 115933, 115934, 115935, 115936, 115937, 115938, 115939, 115940, 115941, 115942, 115943, 115944, 115945, 115946, 115947, 115948, 115949, 115950, 115951, 115952, 115953, 115954, 115955, 115956, 115957, 115958, 115959, 115960, 115961, 115962, 115963, 115964, 115965, 115966, and 115967. A more detailed table can be found in Supplementary Fig. 30. Additional data are available from the corresponding authors upon reasonable request.

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Acknowledgements

The authors would like to thank J. Niles, B.J. Belmont, and S. Maddur Ganesan (MIT) for discussions regarding the TetR–aptamer system, A. Ghodasara (MIT) for discussions and technical assistance related to aptamers/aptazymes, and R. Petersen (Designs By Robin) for consultations with figure design and layout. This work was supported by research grants from the Defense Advanced Research Projects Agency (W911NF-11-2-0054: awarded to J.B., D.D., and R.W., supported T.W., J.B.R., X.Z., E.P., J.B., D.D., T.K., and R.W. and W32P4Q-13-1-0011: awarded to R.W., supported X.Z., T.K., and R.W.), the National Science Foundation (NSF#1522074, NSF#1521759: awarded to D.D., supported T.W. and D.D., CCF-1521925: awarded to D.D. and R.W., supported T.W, J.R.B., D.D., and R.W., CNS-1446607: awarded to R.W., supported J.R.B., B.T., and R.W., and MCB-1745645: awarded to R.W., supported J.R.B. and R.W.), the National Institutes of Health (5-R01-CA206218: awarded to R.W., supported J.B.R., E.P., and R.W.), the Ragon Institute of MGH, MIT and Harvard (awarded to R.W., supported A.W. and R.W.), the Special Research Fund from Ghent University (awarded to N.N.S.), and The Research Foundation - Flanders (FWO; G.0235.11N and G.0621.10N: awarded to N.N.S.). This work was also supported by a sponsored research agreement with Crucell Holland B.V. (awarded to R.W., supported: B.D., E.P., and R.W.). We further acknowledge the following support: MIT-Amgen UROP Scholars Program (K.B.), Gabilan Stanford Graduate Fellowship (K.B.), Fannie and John Hertz Foundation Fellowship - Hertz-Draper Fellow (K.B.), Stanford EDGE-STEM Doctoral Fellowship (K.B.), PhD fellowship and international mobility grant from FWO (O.A.), and the Emmanuel van der Schueren fellowship from “Kom op tegen Kanker” (O.A.).

Author information

Author notes

  1. Katie Bodner

    Present address: Department of Bioengineering, Stanford University, Stanford, CA, USA

  2. Oliwia Andries

    Present address: Perrigo Company PLC, Nazareth, Belgium

  3. Tasuku Kitada

    Present address: Candriam Investors Group, Brussels, Belgium

  4. These authors contributed equally: Tyler E. Wagner, Jacob R. Becraft.

  5. These authors jointly directed this work: Tasuku Kitada, Ron Weiss.

Authors and Affiliations

  1. Department of Biomedical Engineering, Boston University, Boston, MA, USA

    Tyler E. Wagner & Douglas Densmore

  2. Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA

    Tyler E. Wagner, Jacob R. Becraft, Katie Bodner, Brian Teague, Xin Zhang, Amanda Woo, Ely Porter, Brian Dobosh, Tasuku Kitada & Ron Weiss

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jacob R. Becraft, Katie Bodner, Bremy Alburquerque & Ron Weiss

  4. Faculty of Veterinary Medicine, Department of Nutrition, Genetics and Ethology, Laboratory for Gene Therapy, Ghent University, Gent, Belgium

    Oliwia Andries & Niek N. Sanders

  5. Cancer Research Institute Ghent (CRIG), Ghent University, Gent, Belgium

    Niek N. Sanders

  6. Raytheon BBN Technologies, Cambridge, MA, USA

    Jacob Beal

  7. Biological Design Center, Boston University, Boston, MA, USA

    Douglas Densmore

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Contributions

J.R.B., T.W., K.B., and T.K. designed and performed experiments and analyzed data. X.Z., A.W., E.P., and B.D. performed experiments and interpreted data. O.A., N.N.S., and D.D. designed experiments. B.A. performed experiments. J.B. analyzed data. T.K. and R.W. supervised the study. T.W., J.R.B., B.T., T.K., and R.W. wrote the manuscript with the support of all other authors.

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Correspondence to Tasuku Kitada or Ron Weiss.

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Competing interests

MIT has filed a patent application (No. 15/509,258) pertaining to the technology described in this paper. T.W., J.R.B., K.B., T.K., and R.W. are co-inventors on this patent application. J.R.B. and T.K. are shareholders of a company founded based on the technology described in this article.

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Wagner, T.E., Becraft, J.R., Bodner, K. et al. Small-molecule-based regulation of RNA-delivered circuits in mammalian cells. Nat Chem Biol 14, 1043–1050 (2018). https://doi.org/10.1038/s41589-018-0146-9

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