Abstract
Duplication of methyl-CpG-binding protein 2 (MECP2) gene causes MECP2 duplication syndrome (MDS). To normalize the duplicated MECP2 in MDS, we developed a high-fidelity Cas13Y (hfCas13Y) system capable of targeting the MECP2 (hfCas13Y-gMECP2) messenger RNA for degradation and reducing protein levels in the brain of humanized MECP2 transgenic mice. Moreover, the intracerebroventricular adeno-associated virus (AAV) delivery of hfCas13Y-gMECP2 in newborn or adult MDS mice restored dysregulated gene expression and improved behavior deficits. Notably, treatment with AAV9-hfCas13Y-gMECP2 extended the median survival of MECP2 transgenic mice from 156.5 to 226 d. Furthermore, studies with monkeys showed a single injection of AAV9-hfCas13Y-gMECP2 was sufficient to drive robust expression of hfCas13Y in widespread brain regions, with MECP2 knockdown efficiency reaching 52.19 ± 0.03% and significantly decreased expression of biomarker gene GDF11. Our results demonstrate that the RNA-targeting hfCas13Y-gMECP2 system is an effective intervention for MDS, providing a potential strategy for treating other dosage-sensitive diseases.
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Fig. 1: Screening of hfCas13Y variants and efficient gRNAs for MECP2 RNA knockdown.
Fig. 2: MECP2 in vivo knockdown efficiency mediated by the AAV-hfCas13Y RNA editing system.
Fig. 3: Knockdown of MeCP2 by the AAV-PHP.eB-hfCas13Y v3-mediated RNA editing system rescues impaired behaviors of MECP2 TG mice.
Fig. 4: AAV9-mediated delivery of hfCas13Y normalizes MECP2 levels and rescues behavioral deficits in the P1 MECP2 TG mice.
Fig. 5: AAV9-mediated delivery of hfCas13Y v3 via ICV injection normalizes MECP2 levels in a single neuron in the cortex of the MECP2 TG mouse model.
Fig. 6: AAV9-mediated delivery of hfCas13Y-gMECP2 via ICV injection normalized MECP2 levels and mitigated behavioral deficits in 2-month-old MECP2 TG mice.
Fig. 7: AAV9-mediated delivery of hfCas13Y v3 via ICV injection decreased MECP2 levels in the brain of WT male cynomolgus monkeys.
Data availability
Deep-seq data are deposited to the Sequence Read Archive (SRA) database (accession numbers: PRJNA1101724 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1101724), PRJNA1102504 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1102504) and PRJNA1101197 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1101197)), and plasmids are available from the corresponding authors upon request. Source data are provided with this paper.
Code availability
No code was written in the study.
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Acknowledgements
We thank the laboratory animal center at HuidaGene Therapeutics Inc. for technical support. H.Y. was sponsored by the National Natural Science Foundation of China (grant nos. 31925016 and 82021001); the National Science and Technology Innovation 2030 Major Program (grant no. 2021ZD0200900); the Basic Frontier Scientific Research Program of Chinese Academy of Sciences From 0 to 1 original innovation project (grant no. ZDBS-LY-SM001); and the Project of Shanghai Municipal Science and Technology Commission (grant no. 23HC1401100). C.X. was sponsored by the Lingang Laboratory intramural fund and the Shanghai City Committee of Science and Technology Project (grant no. 22QA1412300). X. Wang was sponsored by the China Postdoctoral Science Foundation (grant no. 2023M741325).
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These authors contributed equally: Dong Yang, Xiaoqing Wu, Yinan Yao, Mengsi Duan.
Authors and Affiliations
HuidaGene Therapeutics Inc., Shanghai, China
Dong Yang, Xiaoqing Wu, Mengsi Duan, Xing Wang, Guoling Li, Aiguo Guo, Meixian Wu, Jin Zheng, Tong Li, Alvin Luk, Xuan Yao, Linyu Shi & Hui Yang
Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China
Yinan Yao, Yuanhua Liu, Renxia Zhang & Hui Yang
Lingang Laboratory, Shanghai, China
Chunlong Xu
Shanghai Research Center for Brain Science and Brain-Inspired Technology, Shanghai, China
Chunlong Xu & Hui Yang
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Dong Yang
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Contributions
D.Y., L.S., C.X. and H.Y. conceived the project and experiments. D.Y., X. Wu, Y.Y., M.D., X. Wang, G.L., A.G., M.W., J.Z. and R.Z. performed the experiments and analyzed the data. Y.L. performed the bioinformatic analyses. D.Y., Y.Y., C.X., A.L., T.L. and H.Y. wrote the manuscript with input from all authors. H.Y. and X.Y. supervised research and acquired funding.
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Correspondence to Xuan Yao, Linyu Shi, Chunlong Xu or Hui Yang.
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H.Y. is the founder of HuidaGene Therapeutics Inc. The other authors declare no competing interests.
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Nature Neuroscience thanks Bence György, Huda Zoghbi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Supplementary Figs. 1–17, Tables 1–5, Results 1–3, Discussion, Methods and References.
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Supplementary Data 1. Statistical source data and unprocessed western blot for the supplementary figures.
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Source Data Fig. 1. Statistical source data and unprocessed western blot. Source Data Fig. 2. Statistical source data and unprocessed western blot. Source Data Fig. 3. Statistical source data. Source Data Fig. 4. Statistical source data. Source Data Fig. 5. Statistical source data. Source Data Fig. 6. Statistical source data and unprocessed western blot. Source Data Fig. 7. Statistical source data and unprocessed western blot.
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Yang, D., Wu, X., Yao, Y. et al. An RNA editing strategy rescues gene duplication in a mouse model of MECP2 duplication syndrome and nonhuman primates. Nat Neurosci (2024). https://doi.org/10.1038/s41593-024-01838-6
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Received:27 July 2023
Accepted:24 October 2024
Published:12 December 2024
DOI:https://doi.org/10.1038/s41593-024-01838-6
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