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CRISPR/Cas9-mediated exon skipping to restore premature translation termination in a DFNB4 mouse model

Gene Therapy volume 31, pages 531–540 (2024)Cite this article

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Abstract

SLC26A4 encodes pendrin, a crucial anion exchanger essential for maintaining hearing function. Mutations in SLC26A4, including the prevalent c.919-2 A > G splice-site mutation among East Asian individuals, can disrupt inner ear electrolyte balance, leading to syndromic and non-syndromic hearing loss, such as Pendred syndrome and DFNB4. To explore potential therapeutic strategies, we utilized CRISPR/Cas9-mediated exon skipping to create a Slc26a4∆E8+E9/∆E8+E9 mouse model. We assessed pendrin expression in the inner ear and evaluated vestibular and auditory functions. The Slc26a4∆E8+E9/∆E8+E9 mice demonstrated reframed pendrin in the inner ear and normal vestibular functions, contrasting with severely abnormal vestibular functions observed in the Slc26a4 c.919-2 A > G splicing mutation mouse model. However, despite these molecular achievements, hearing function did not show the expected improvement, consistent with observed pathology, including cochlear hair cell loss and elevated hearing thresholds. Consequently, our findings highlight the necessity for alternative genetic editing strategies to address hearing loss caused by the SLC26A4 c.919-2 A > G mutation.

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Fig. 1: CRISPR/Cas9-mediated deletion in the exons 8 and 9 of Slc26a4 gene in Neuro-2a cells.
Fig. 2: Detection of reframed Slc26a4 transcripts in the Slc26a4∆E8+E9/∆E8+E9 mice.
Fig. 3: Reframed pendrin expression in the endolymphatic sac of Slc26a4∆E8+E9/∆E8+E9 mice.
Fig. 4: Slc26a4∆E8+E9/∆E8+E9 mice exhibit normal locomotor behaviors.
Fig. 5: Elevated hearing threshold with cochlear hair cell loss observed in the Slc26a4∆E8+E9/∆E8+E9 mice.
Fig. 6: Structural model of pendrin highlighting the central role of exons 8 and 9 in the pore formation.
Fig. 7: Topology of pendrins.

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All the data produced in this study have been either included in the published paper or are accessible through the lead contact upon request.

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Acknowledgements

We thank the National Laboratory Animal Center (NLAC), NARLabs, Taiwan, for technical support in contract breeding and testing services. We are also grateful to Dr. Yu-Chi Chou in the RNA Technology Platform and Gene Manipulation Core Facility (RNAi core) of the National Core Facility for Biopharmaceuticals at Academia Sinica in Taiwan for providing CRISPR reagents and related services. This work was supported by grants from the National Science and Technology Council (NSTC 111-2314-B-A49A-504-MY3), the Taipei Veterans General Hospital (V110C-123, V112C-141), the National Health Research Institutes (NHRI-EX112-11005NI), and the Taipei Veterans General Hospital-National Taiwan University Hospital Joint Research Program (VN111-16, VN112-08).

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Author notes

  1. These authors contributed equally: Chun-Ying Huang, Yi-Hsiu Tsai, Yi-Fen Cheng.

Authors and Affiliations

  1. Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan

    Chun-Ying Huang, Yi-Fen Cheng, Po-Yuan Huang & Yen-Fu Cheng

  2. School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan

    Chun-Ying Huang & Yen-Fu Cheng

  3. Institute of Brain Science, National Yang Ming Chiao Tung University, Taipei, Taiwan

    Yi-Hsiu Tsai, Peng-Yu Wu, Yu-Chi Chuang & Yen-Fu Cheng

  4. Department of Biotechnology and Pharmaceutical Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan

    Jai-Shin Liu

  5. Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan

    Chen-Chi Wu

  6. Department of Medical Research, National Taiwan University Hospital Hsin-Chu Branch, Hsin-Chu City, Taiwan

    Chen-Chi Wu

  7. Department of Otolaryngology-Head and Neck Surgery, Taipei Veterans General Hospital, Taipei, Taiwan

    Yen-Fu Cheng

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Contributions

YFC and CCW contributed to the study design. CYH and YFC contributed to the mouse model design. PTW, PYH, and YCC contributed to mouse breeding and genotyping. YHT and PTW contributed to fluorescence and H&E staining. YFC and PTW contributed to vestibular function. JSL and YCC contributed to protein structure modeling. CYH, YHT, YFC, and YFC contributed to the writing of the manuscript. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Chen-Chi Wu or Yen-Fu Cheng.

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Huang, CY., Tsai, YH., Cheng, YF. et al. CRISPR/Cas9-mediated exon skipping to restore premature translation termination in a DFNB4 mouse model. Gene Ther 31, 531–540 (2024). https://doi.org/10.1038/s41434-024-00483-9

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