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Control of mammalian gene expression by modulation of polyA signal cleavage at 5′ UTR

Nature Biotechnology (2024)Cite this article

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Abstract

The ability to control gene expression in mammalian cells is crucial for safe and efficacious gene therapies and for elucidating gene functions. Current gene regulation systems have limitations such as harmful immune responses or low efficiency. We describe the pA regulator, an RNA-based switch that controls mammalian gene expression through modulation of a synthetic polyA signal (PAS) cleavage introduced into the 5′ UTR of a transgene. The cleavage is modulated by a ‘dual-mechanism’—(1) aptamer clamping to inhibit PAS cleavage and (2) drug-induced alternative splicing that removes the PAS, both activated by drug binding. This RNA-based methodology circumvents the immune responses observed in other systems and achieves a 900-fold induction with an EC50 of 0.5 µg ml−1 tetracycline (Tc), which is well within the FDA-approved dose range. The pA regulator effectively controls the luciferase transgene in live mice and the endogenous CD133 gene in human cells, in a dose-dependent and reversible manner with long-term stability.

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Fig. 1: Modulation of PAS-mediated cleavage via Tc-binding aptamer.
Fig. 2: The Y-shaped structure converges the binding forces from three aptamers.
Fig. 3: Controlling PAS by alternative splicing as a second mechanism.
Fig. 4: Evaluation of the pA regulator in vitro.
Fig. 5: In vivo evaluation of the pA regulator.
Fig. 6: Controlling the expression of endogenous CD133 gene in human genome by the pA regulator.

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Data availability

The data that support the findings of this study are included in the manuscript and its Supplementary information file. Source data are provided with this paper.

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Acknowledgements

The authors would like to thank T. Cooper and R. Sifers for their critical suggestions, and the Gene Vector Core at Baylor College of Medicine and K. Oka for consultation and AAV production. J.D.-Y.J. was supported by E&M Foundation Pre-Doctoral Fellowship for Biomedical Research. L.Y. was supported by NIH R01EB013584, Biogen SRA and the seed fund from the Department of Pathology and Immunology, Baylor College of Medicine. This project was also supported in part by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (P30 CA125123 and S10 RR024574) and the expert assistance of J.M. Sederstrom. The authors would also like to thank C. Ward and the Mouse Metabolism and Phenotyping Core at Baylor College of Medicine for expert assistance and funding from NIH UM1HG006348, NIH R01DK114356 and NIH R01HL130249.

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  1. These authors contributed equally: Liming Luo, Jocelyn Duen-Ya Jea.

Authors and Affiliations

  1. Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA

    Liming Luo, Jocelyn Duen-Ya Jea, Yan Wang, Pei-Wen Chao & Laising Yen

  2. Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA

    Liming Luo, Jocelyn Duen-Ya Jea, Yan Wang, Pei-Wen Chao & Laising Yen

  3. Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Liming Luo, Jocelyn Duen-Ya Jea, Yan Wang, Pei-Wen Chao & Laising Yen

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Contributions

L.L., J.D.-Y.J., Y.W. and P.-W.C. performed experiments. L.Y. conceived the project and obtained the funding. L.L., J.D.-Y.J. and L.Y. wrote the manuscript.

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Correspondence to Laising Yen.

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L.Y. was partially supported by a ‘Sponsored Research Agreement’ from Biogen. L.Y., J.D.-Y.J., L.L. and P.-W.C. received patent loyalty from Biogen (patents WO2017083747 and WO2021041924A2). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Luo, L., Jea, J.DY., Wang, Y. et al. Control of mammalian gene expression by modulation of polyA signal cleavage at 5′ UTR. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-01989-0

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