Abstract
Measurement of gene expression in the brain requires invasive analysis of brain tissue or non-invasive methods that are limited by low sensitivity. Here we introduce a method for non-invasive, multiplexed, site-specific monitoring of endogenous gene or transgene expression in the brain through engineered reporters called released markers of activity (RMAs). RMAs consist of an easily detectable reporter and a receptor-binding domain that enables transcytosis across the brain endothelium. RMAs are expressed in the brain but exit into the blood, where they can be easily measured. We show that expressing RMAs at a single mouse brain site representing approximately 1% of the brain volume provides up to a 100,000-fold signal increase over the baseline. Expression of RMAs in tens to hundreds of neurons is sufficient for their reliable detection. We demonstrate that chemogenetic activation of cells expressing Fos-responsive RMA increases serum RMA levels >6-fold compared to non-activated controls. RMAs provide a non-invasive method for repeatable, multiplexed monitoring of gene expression in the intact animal brain.
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Data availability
The authors declare that all data supporting the results in this study are available within the paper, its Supplementary Information and its Source Data file. Microscopy images are available from the corresponding author upon reasonable request owing to their large size and number. The plasmids designed in this study are available on Addgene (https://www.addgene.org/browse/article/28229133/). Source data are provided with this paper.
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Acknowledgements
We thank J. J. Tabor (Rice University) for providing the pET28a bacterial expression plasmid and G. Bao (Rice University) for providing use of the ultracentrifuge. We thank V. Gradinaru (Caltech) and the Caltech CLOVER Center for providing the pUCmini-iCAP-PHP.eB and pHelper plasmids. This research was supported by the David and Lucile Packard Foundation 2021-73005 (J.O.S.), National Institute of Biomedical Imaging and Bioengineering Trailblazer Award R21EB033059 (J.O.S.), National Institute of General Medical Sciences DP2GM140923 (G.C.), National Institute on Drug Abuse R00DA043609 (G.C.) and National Institute of Neurological Disorders and Stroke F31NS125927 (J.A.W.).
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J.O.S. and S.L. conceived and planned the research. S.L. and J.O.S. designed the experiments and wrote the paper, with input from all other authors. S.L. performed and participated in all experiments described in the study. S.N. performed AAV production. S.N. and J.A.W. performed stereotaxic injection and retro-orbital blood collection. S.N. and J.J.K. performed the histological experiments. J.J.K. maintained PC-12 and conducted the in vitro Fos activation. S.N., J.A.W., J.J.K., Z.H. and B.X. analyzed histological images. S.N. and Z.H. assisted with IVIS imaging and drug administration. Z.H. constructed plasmids for the chemogenetic experiment. B.X. maintained and transfected astrocytes and performed protein purification. G.C. provided advice on the chemogenetic experiment and experimental guidance to J.A.W.
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J.O.S. and S.L. are co-inventors on a US patent application that incorporates discoveries described in this paper. The other authors declare no competing interests.
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Lee, S., Nouraein, S., Kwon, J.J. et al. Engineered serum markers for non-invasive monitoring of gene expression in the brain. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-02087-x
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DOI: https://doi.org/10.1038/s41587-023-02087-x