Postnatal Slc26a4 gene therapy improves hearing and structural integrity in a hereditary hearing loss model
Yi-Hsiu Tsai, Peng-Yu Wu, Yu-Chi Chuang, Chun-Ying Huang, Hiroki Takeda, Hiroshi Hibino, Chen-Chi Wu, Yen-Fu Cheng
Yi-Hsiu Tsai, Peng-Yu Wu, Yu-Chi Chuang, Chun-Ying Huang, Hiroki Takeda, Hiroshi Hibino, Chen-Chi Wu, Yen-Fu Cheng
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Research Article Genetics Otology

Postnatal Slc26a4 gene therapy improves hearing and structural integrity in a hereditary hearing loss model

Abstract

Mutations in SLC26A4 are the second most common cause of hereditary hearing loss (HL) in many Asian countries, leading to DFNB4, a condition characterized by progressive HL and inner ear malformations. While gene therapy holds great potential, its postnatal application has remained unexplored because of the lack of suitable animal models and the challenges of prenatal intervention. To our knowledge, this study represents the first preclinical investigation of postnatal gene therapy for DFNB4 using a clinically relevant Slc26a4-mutant mouse model that closely replicates human auditory phenotypes. Utilizing the synthetic AAV.Anc80L65 vector, we achieved robust SLC26A4 delivery to critical cochlear regions, including the endolymphatic sac and cochlear lateral wall. Comprehensive phenotypic analyses revealed a critical therapeutic window spanning the neonatal and juvenile stages, within which AAV.Anc80L65-mediated SLC26A4 delivery significantly improved hearing, as evidenced by lower auditory brainstem response thresholds. Moreover, the therapy preserved hair cells, reduced endolymphatic sac enlargement, partially restored the endocochlear potential, and mitigated inner ear structural degeneration. These therapeutic effects persisted into adulthood, highlighting the long-term efficacy of postnatal gene therapy. Together, these findings establish a critical therapeutic window for DFNB4 and demonstrate the feasibility of targeting the endolymphatic sac and cochlear lateral wall for effective intervention.

Authors

Yi-Hsiu Tsai, Peng-Yu Wu, Yu-Chi Chuang, Chun-Ying Huang, Hiroki Takeda, Hiroshi Hibino, Chen-Chi Wu, Yen-Fu Cheng

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Figure 2

The AAV.Anc80L65 vector effectively transduces the endolymphatic sac and lateral wall cells without affecting hearing in WT mice.

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The AAV.Anc80L65 vector effectively transduces the endolymphatic sac and...
(A) Experimental timeline of neonatal delivery of Anc80.GFP and Anc80.hSLC26A4 to WT mice. The illustration was created with BioRender.com. (B and C) The transduction tropism of Anc80.GFP in the endolymphatic sac of WT mice. Original magnification of confocal images, ×10 (B) and ×40 (C, zoomed-in white square area in B). The green, gray, red, and blue channels represent GFP, pendrin, phalloidin, and DAPI, respectively. Scale bars: 100 μm (B) and 10 μm (C). (D) Transduction tropism of Anc80.GFP in spiral prominence cells in the lateral wall of WT mice. The green, gray, red, and blue channels represent GFP, pendrin, phalloidin, and DAPI, respectively. Scale bars: 50 μm. (E) Quantification of the images in C. Data are presented as the mean ± SD (n = 11 AAV-injected mice). ***P < 0.001, by unpaired, 2-tailed t test. (F) Quantification of the images in D. Data are presented as the mean ± SD (n = 11 AAV injected mice). (G) Schematic diagram of the transgene construct. The full-length hSLC26A4 coding sequence and FLAG sequence were driven by a CAG promoter (CMV enhancer and CBA promoter), followed by the bovine growth hormone (bGH) polyA sequence, flanked by AAV2 ITR, and packaged into an AAV.Anc80L65 capsid. (H) ABR waveforms of WT mice injected by Anc80.hSLC26A4 (left panel) and vehicle (right panel) under 32 kHz stimulation at P30. The threshold is represented in bold lines in each panel. (I) Mouse ABR thresholds under click, 8, 16, and 32 kHz stimulations. Data are presented as the mean ± SD (n = 8 injected mice in each group).