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Gene therapy restores auditory function and rescues damaged inner hair cells in an aged Vglut3 knockout mouse model

Gene Therapy (2025)Cite this article

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Abstract

Vesicular glutamate transporter 3 (VGLUT3) is prominently expressed in the inner hair cells of the cochlea, playing a vital role in auditory signal transmission to the brain. Previous studies have shown that Vglut3 gene knockout in mice causes severe sensorineural hearing loss without affecting hair cell integrity. However, the cochlear structure of the aged Vglut3KO remains inadequately explored. In this study, we analyzed the cochlear structure of aged Vglut3KO mice, revealing significant degeneration of inner hair cells, synapses, and stereocilia. To explore the potential of gene therapy to restore cochlear structure, we employed AAV8 vectors to express Vglut3 in the cochleae of 5-week-old Vglut3KO mice. Twenty-seven weeks post-injection, we conducted a series of experiments to evaluate the efficacy of our gene therapy approach. Auditory brainstem response (ABR) testing demonstrated restoration of auditory function following gene therapy. Immunohistochemical staining and scanning electron microscopy (SEM) analysis revealed substantial recovery of inner hair cells and stereocilia post-injection. Our findings provide important insights into the development of novel therapeutic strategies for age-related hearing loss.

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Fig. 1: Hair cell counts in the cochleae of Vglut3WT and Vglut3KO mice at 5, 20, and 32 weeks of age.
Fig. 2: Synaptic ribbons counts in the cochleae of Vglut3WT and Vglut3KO mice at 5, 20, and 32 weeks of age.
Fig. 3: Spiral ganglion neuron counts and nerve fiber bundle widths in Vglut3WT and Vglut3KO mice at 7 and 32 weeks of age.
Fig. 4: AAV8 restored Vglut3 expression in the cochleae of 5-week-old Vglut3KO mice.
Fig. 5: AAV8 restores the stereocilia of hair cells in the middle turn of cochlea in 32-week-old Vglut3KO mice.
Fig. 6: Recovery of auditory function in 5-week-old Vglut3KO mice following AAV8-mediated Vglut3 gene transfer.
Fig. 7: Expression of Vglut3 in the cochlea of Vglut3KO mice at 2, 12, and 27 weeks post-treatment.

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Data availability

The data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the China Postdoctoral Science Foundation (2024M752977), the Medical Science and Technology Research Plan Joint Construction Project of Henan Province (LHGJ20240279), the National Natural Science Foundation of China (82371160), and the Postdoctoral Research Foundation of the First Affiliated Hospital of Zhengzhou University (72137). We also thank the Precision Medicine Center, Academy of Medical Sciences, Zhengzhou University, and the Key Laboratory for Research on Deafness Mechanism, Henan Provincial Health Commission (Henan Health Science and Education Letter 2021, No. 44) for their support.

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  1. These authors contributed equally: Xingle Zhao, Hongen Xu, Chengyu Lian

Authors and Affiliations

  1. Department of Otolaryngology Head and Neck Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

    Xingle Zhao, Shousen Hu, Jia Wang, Rongqun Zhai, Mihuan Yang, Wei Lu & Liang Wang

  2. Precision Medicine Center, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, China

    Hongen Xu, Chengyu Lian & Yue Zhao

  3. The Research and Application Center of Precision Medicine, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, China

    Hongen Xu & Wenxue Tang

  4. Department of Cardiology, The 7th People’s Hospital of Zhengzhou, Zhengzhou, China

    Yuanjing Zhang

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Contributions

LW initiated and managed the project, while LW and WT conceived and designed the experiments. XZ, HX, and CL performed most of the experiments and data analysis. All authors contributed to data analysis, interpretation, and presentation. XZ and HX wrote the manuscript with contributions from all authors. The supplementary experiments to answer the reviewer’s questions were mainly completed by WL, XZ and RZ.

Corresponding authors

Correspondence to Wei Lu, Wenxue Tang or Liang Wang.

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The authors declare no competing interests.

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All methods were performed in accordance with the relevant guidelines and regulations. All animals were bred in accredited barrier facilities with a controlled environment, feed, and water. The mice were housed in a room with a 12-h light/12-h dark cycle, and both water and food were provided ad libitum. All relevant research was approved by the Institutional Animal Care and Use Committee of Zhengzhou University (Ethics ID: ZZU-LAC20230526 (08)). All experiments were conducted in accordance with the 3Rs principles of animal research: Reduction, Replacement, and Refinement. Animals exhibiting severe health impairment were systematically excluded from analysis based on the following objective indicators: weight loss exceeding 20% of baseline body weight. Informed consent for publication was obtained from all participants.

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Zhao, X., Xu, H., Lian, C. et al. Gene therapy restores auditory function and rescues damaged inner hair cells in an aged Vglut3 knockout mouse model. Gene Ther (2025). https://doi.org/10.1038/s41434-025-00558-1

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