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Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression

Nature Biotechnology volume 40, pages 1143–1149 (2022)Cite this article

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Abstract

Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes (‘reporter genes’) that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides (‘reporter probes’). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals.

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Fig. 1: MRI pseudocolor reporter system.
Fig. 2: Design of highly active and orthogonal Dm-dNK and HSV1-TK reporter genes.
Fig. 3: In vitro and in vivo MRI of GeneREFORM.

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Data availability

The datasets generated in this study are available at https://doi.org/10.5281/zenodo.5594956. Source data are provided with this paper.

Code availability

The PROSS-design algorithm is available for noncommercial use through a webserver online (http://pross.weizmann.ac.il). The MATLAB scripts used for processing the CEST data are available at http://godzilla.kennedykrieger.org/CEST/. The Python code and the relevant data used for CEST simulations can be found at https://github.com/nirbhayyadav/NBT-RA51532A.

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Acknowledgements

This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement nos. 677715 and 815379 to A.B.-S. and S.J.F., respectively) and by a charitable donation in memory of Sam Switzer (to S.J.F.).

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Authors and Affiliations

  1. Department of Molecular Chemistry and Materials Science, Weizmann Institute of Science, Rehovot, Israel

    Hyla Allouche-Arnon, Nishanth D. Tirukoti & Amnon Bar-Shir

  2. Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel

    Olga Khersonsky & Sarel J. Fleishman

  3. Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot, Israel

    Yoav Peleg, Orly Dym, Shira Albeck, Alexander Brandis & Tevie Mehlman

  4. Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

    Liat Avram & Talia Harris

  5. Russell H. Morgan Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Nirbhay N. Yadav

  6. F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, Baltimore, MD, USA

    Nirbhay N. Yadav

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  1. Hyla Allouche-Arnon
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Contributions

H.A.-A. and A.B.-S. designed the study. O.K. and S.J.F. performed and analyzed computational design calculations. N.D.T. synthesized 5-MDHT. H.A.-A. carried out all experiments, including cloning, transfections, cell-line establishment, western blots, FACS and in vivo CEST experiments, tumor inoculations, AAV viral infections and histology. Y.P., O.D. and S.A. performed bacterial protein expression, purification and crystallization. A.B. and T.M. developed optimized and performed LC–MS experiments. H.A.-A. and L.A. performed NMR experiments and H.A.-A. and T.H. performed in vitro CEST experiments. N.N.Y. performed CEST simulations and postprocessing of in vitro CEST data using Lorentzian line fitting approach. H.A.-A. and A.B.-S. analyzed the data and wrote the paper with input from O.K. and S.J.F.

Corresponding author

Correspondence to Amnon Bar-Shir.

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Nature Biotechnology thanks Kevin Brindle, Franz Schilling and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary protein sequences, Methods, Figs. 1–21, Tables 1–8 and References.

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Source Data Fig. 2

Unprocessed western blots for Fig. 2a,j.

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Statistical source data Fig. 2.

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Statistical source data Fig. 3.

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Allouche-Arnon, H., Khersonsky, O., Tirukoti, N.D. et al. Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression. Nat Biotechnol 40, 1143–1149 (2022). https://doi.org/10.1038/s41587-021-01162-5

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