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AAV-Net1 facilitates the trans-differentiation of supporting cells into hair cells in the murine cochlea

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Abstract

Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFPCreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFPCreERT2/Rosa26-tdTomatoloxp/loxp mice. We further found that the up-regulation of Wnt/β-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.

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Funding

This work was supported by the National Key Research and Development Program of China (2021YFA1101300, 2020YFA0113600, 2021YFA1101800 and 2020YFA0112503), the Strategic Priority Research Program of the Chinese Academy of Science (XDA16010303), the National Natural Science Foundation of China (82000984 , 92149304, 82030029 and 81970882), the China National Postdoctoral Program for Innovative Talents (BX20200082), the China Postdoctoral Science Foundation (2020M681468), the Science and Technology Department of Sichuan Province (2021YFS0371), the Shenzhen Fundamental Research Program (JCYJ20190814093401920 and JCYJ20210324125608022), and the Open Research Fund of State Key Laboratory of Genetic Engineering, Fudan University (SKLGE-2104).

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Author notes

  1. Liyan Zhang, Yuan Fang, Fangzhi Tan and Fangfang Guo contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Bioelectronics, Department of Otolaryngology Head and Neck Surgery, Zhongda Hospital, School of Life Sciences and Technology, Advanced Institute for Life and Health, Jiangsu Province High-Tech Key Laboratory for Bio-Medical Research, Southeast University, Nan**g, 210096, China

    Liyan Zhang, Yuan Fang, Fangzhi Tan, Ziyu Zhang, Nianci Li, Qiuhan Sun, Jieyu Qi & Renjie Chai

  2. Department of Plastic and Reconstruction Surgery, Zhongda Hospital, Southeast University, 87 Dingjiaqiao Street, Nan**g, Jiangsu Province, China

    Fangfang Guo

  3. Department of Otolaryngology Head and Neck Surgery, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China

    Renjie Chai

  4. Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, 226001, China

    Renjie Chai

  5. Institute for Stem Cell and Regeneration, Chinese Academy of Science, Bei**g, China

    Renjie Chai

  6. Bei**g Key Laboratory of Neural Regeneration and Repair, Capital Medical University, Bei**g, 100069, China

    Renjie Chai

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  1. Liyan Zhang
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Contributions

JQ and RC conceived and designed the experiments. LZ, YF, and FT performed most of the experiments. LZ contributed to the data analysis. FG completed the ABR test and data analyses of RNA sequencing. ZZ, NL, and QS helped with the experiments and the data analysis. JQ, LZ, YF, and FT discussed the data analysis, interpretation, and presentation and wrote the manuscript with contributions from all authors.

Corresponding authors

Correspondence to Jieyu Qi or Renjie Chai.

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Conflict of interest

Jieyu Qi had filed a patent on the use of AAV-ie for gene therapy in the inner ear. The authors declare no other competing interests.

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Zhang, L., Fang, Y., Tan, F. et al. AAV-Net1 facilitates the trans-differentiation of supporting cells into hair cells in the murine cochlea. Cell. Mol. Life Sci. 80, 86 (2023). https://doi.org/10.1007/s00018-023-04743-6

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