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Ptbp1 knockdown failed to induce astrocytes to neurons in vivo

Gene Therapy (2023)Cite this article

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Abstract

The conversion of non-neuronal cells to neurons is a promising potential strategy for the treatment of neurodegenerative diseases. Recent studies have reported that shRNA-, CasRx-, or ASO-mediated Ptbp1 suppression could reprogram resident astrocytes to neurons. However, some groups have disputed the interpretation of the data underlying the reported neuron conversion events. These controversies surrounding neuron conversion may be due to differences in the astrocyte fate-mapping systems. Here, we suppressed Ptbp1 using Cas13X and labelled astrocytes with an HA tag fused to Cas13X (Cas13X-NLS-HA). We found no astrocyte-to-neuron conversion in the mouse striatum via the HA-tagged labelling system compared with the GFAP-driven tdTomato labelling system (AAV-GFAP::tdTomato-WPRE) used in previous studies. Our findings indicate that Cas13X-mediated Ptbp1 knockdown failed to induce neuron conversion in vivo.

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Fig. 1: Ptbp1-knockdown by Cas13X and sgRNA in vitro and in vivo.
Fig. 2: Astrocyte-to-Neuron conversion after Ptbp1 knockdown validated by tdTomato mediated astrocyte-labeling system.
Fig. 3: No astrocyte-to-Neuron conversion occurred after Ptbp1 knockdown validated by HA mediated astrocyte labeling system.

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Acknowledgements

We thank Weiya Bai and Guannan Geng for AAV vector preparation, Yanxia Gao and Leping Cheng for project discussion.

Funding

This work was supported by National Science and Technology Innovation 2030 Major Program (2021ZD0200900); Lingang Laboratory (LG202106-01-02); Strategic Priority Research Program of Chinese Academy of Sciences (XDB32060000); National Natural Science Foundation of China (31925016, 82021001); Basic Frontier Scientific Research Program of Chinese Academy of Sciences From 0 to 1 original innovation project (ZDBS-LY-SM001); Shanghai Municipal Science and Technology Major Project (2018SHZDZX05); Project of Shanghai Municipal Science and Technology Commission (20MC1920400).

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Author notes

  1. These authors contributed equally: Guixiang Yang, Zixiang Yan.

Authors and Affiliations

  1. Huigene Therapeutics Inc., Shanghai, China

    Guixiang Yang, Xiaoqing Wu, Meng Zhang, Linyu Shi & Hui Yang

  2. Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Zixiang Yan, Hui Yang & Kailun Fang

  3. Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China

    Zixiang Yan

  4. Lingang Laboratory/Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai, China

    Chunlong Xu

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Contributions

LS, HY, and KF designed the research, GY, ZY, XW, and MZ performed experiments and analyzed data. CX, HY, and KF wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Hui Yang or Kailun Fang.

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Competing interests

HY and LS are co-founders of HuiGene Therapeutics Co., Ltd. GY, XW and MZ are employees of HuiGene Therapeutics Co. The remaining authors declare no conflict of interest.

Ethical approval

This study was approved by Animal Care and Use Committee of the Institute of Huigene Therapeutics Inc., Shanghai, China. All the procedure was performed with sterile, clinical-grade surgical instruments and by experienced technicians.

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Yang, G., Yan, Z., Wu, X. et al. Ptbp1 knockdown failed to induce astrocytes to neurons in vivo. Gene Ther (2023). https://doi.org/10.1038/s41434-023-00382-5

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  • DOI: https://doi.org/10.1038/s41434-023-00382-5

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