Synthetic dosage-compensating miRNA circuits allow precision gene therapy for Rett syndrome
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1.
California Institute of Technology
Abstract
A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight therapeutic window. For example, loss of MECP2 function causes Rett syndrome, while its duplication causes MECP2 duplication syndrome. Viral gene delivery methods generate variable numbers of gene copies in individual cells, creating a need for gene dosage-invariant expression systems. Here, we introduce a compact miRNA-based, incoherent feed-forward loop circuit that achieves precise control of Mecp2 expression in cells and brains, and improves outcomes in an AAV-based mouse model of Rett syndrome gene therapy. Single molecule analysis of endogenous and ectopic Mecp2 mRNA revealed precise, sustained expression across a broad range of gene dosages. Delivered systemically in a brain-targeting AAV capsid, the circuit strongly suppressed Rett behavioral symptoms for over 24 weeks, outperforming an unregulated gene therapy. These results demonstrate that synthetic miRNA-based regulatory circuits can enable precise in vivo expression to improve the safety and efficacy of gene therapy.
Copyright and License
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Acknowledgement
We thank Mitch Gutman for informing us about the overexpression problem for Rett syndrome gene therapy, Inna Strazhnik for graphics, Rana Eser for assistance in tissue handling and animal collections, Martin Tran and Yodai Takei for advice on smFISH and HCR experiments and data analysis, and members of the Elowitz and Gradinaru labs for other input and feedback.
Funding
This work was funded by grants from the Rett Syndrome Research Trust, the Rosen Bioengineering Center, and the Merkin Institute for Translational Research. Research reported in this publication was also supported by the National Institute Of Biomedical Imaging And Bioengineering of the National Institutes of Health under Award Number R01EB030015 and NIH Pioneer DP1OD025535 and U24MH131054. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Contributions
M.J.F. and M.B.E. conceived the project. M.J.F. identified the incoherent-feedforward-loop motif, performed mathematical modeling, and implemented the circuit using miRNA inside an AAV gene expression cassette. M.J.F. validated the circuit in cell culture using flow cytometry, smFISH, and HCR, wrote the image analysis pipeline, and compared endogenous and ectopic expression of MeCP2 in the mouse brain. M.B.E. provided guidance in circuit design and data analysis. A.M.M. and V.G. selected viral capsids and designed the mouse behavioral study. A.M.M. prepped and injected virus, and performed mouse tissue preparation. A.M.M established Rett model mouse colonies, and performed mouse behavioral studies, including time-course of behavioral phenotyping. R.D. performed mRNA sequencing in response to miRNA expression. M.J.F. and M.B.E. wrote the manuscript with input from all authors. M.B.E. supervised the in vitro work and V.G. supervised the in vivo work. M.B.E. and V.G. funded the project.
Conflict of Interest
A patent has been filed by the California Institute of Technology related to this work (US application number 17/100,857). M.B.E. is a scientific advisory board member or consultant at TeraCyte, Primordium, and Spatial Genomics.
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Additional details
- PMCID
- PMC10980028
- Rett Syndrome Research Trust
- Donna and Benjamin M. Rosen Bioengineering Center
- California Institute of Technology
- Merkin Institute for Translational Research
- California Institute of Technology
- Caltech groups
- Rosen Bioengineering Center, Division of Biology and Biological Engineering, Richard N. Merkin Institute for Translational Research, Tianqiao and Chrissy Chen Institute for Neuroscience