Deciphering Enhancers of Hearing Loss Genes for Efficient and Targeted Gene Therapy of Hereditary Deafness

Neurology Bioengineering Genetics Life Sciences Medicine
hearing loss gene therapy hereditary deafness enhancers targeted therapy

Academic Background

Hereditary deafness is one of the most common sensory disorders worldwide, affecting over 400 million people, with approximately 60% of congenital deafness linked to genetic factors. Although adeno-associated virus (AAV)-mediated gene therapy shows great potential in treating hereditary deafness, significant concerns remain regarding its specificity and safety. The complexity of the cochlear structure further increases the challenge of precise gene delivery. To address these issues, researchers have developed a new workflow—AAV-reporter-based in vivo transcriptional enhancer reconstruction (ARBITER)—to dissect enhancers of hearing loss genes. This study aims to achieve efficient and targeted gene therapy by identifying and engineering enhancers, thereby restoring hearing function.

Source of the Paper

This paper was co-authored by Simeng Zhao, Qiuxiang Yang, Zehua Yu, and others, from the iHuman Institute of ShanghaiTech University, the School of Life Science and Technology, and the Kunming Institute of Zoology, Chinese Academy of Sciences, among other institutions. The paper was published on May 21, 2025, in the journal Neuron, titled Deciphering Enhancers of Hearing Loss Genes for Efficient and Targeted Gene Therapy of Hereditary Deafness.

Research Process

1. Establishment of the ARBITER Workflow

The research team first established the ARBITER workflow, which utilizes the AAV-IE vector to deliver reporter genes containing synthetic gene regulatory elements to the cochlea. If these elements are indeed necessary for gene expression, the reporter gene carried by the AAV vector will exhibit a similar expression pattern to the target gene. Researchers delivered the AAV-IE reporter gene to the cochleae of neonatal mice via round window injection (RWI) and collected tissues two weeks later for immunofluorescence staining. The results showed that the AAV-IE vector combined with the CAG promoter efficiently transduced cochlear cells, with nearly 100% of hair cells (HCs) and 80%-95% of supporting cells (SCs) being transduced.

2. Identification and Validation of Enhancers

By analyzing previously published ATAC-seq and ChIP-seq datasets, researchers identified open chromatin sequences and transcription factor binding sites near the target genes. Additionally, they analyzed conserved non-coding elements (CNEs) within the gene loci using a genome browser and selected CNEs longer than 100 bp as candidate enhancers. Based on these elements, researchers designed a series of reporter gene constructs and delivered them to the cochleae of neonatal mice via the AAV-IE vector. The results showed that two CNEs within the SLC26A5 gene locus (SLC26A5-e1 and e2) could collaboratively regulate the expression of SLC26A5.

3. Functional Validation of Enhancers

To validate the function of these enhancers, researchers created SLC26A5-e1 knockout mice and SLC26A5-e1 + e2 double knockout mice using CRISPR-Cas9-mediated gene editing. Phenotypic characterization revealed that SLC26A5-e1 knockout led to a progressive reduction in prestin expression during maturation, while SLC26A5-e1 + e2 double knockout completely abolished prestin expression, resulting in severe deafness.

4. Engineering and Optimization of Enhancers

To overcome the low expression efficiency of the SLC26A5-e1 + e2 enhancer, researchers optimized the enhancer using the ARBITER workflow. They divided the e1 and e2 enhancers into small modules and designed a series of synthetic enhancers. The results showed that synthetic enhancers combining e1p3 and e2p2 or e2p3 modules (such as b8) significantly improved gene expression efficiency. Researchers further evaluated the therapeutic effect of the b8 enhancer in SLC26A5 knockout mice, and the results demonstrated that b8-mediated SLC26A5 delivery efficiently restored hearing function in the mice.

Key Results

  1. Establishment of the ARBITER Workflow: The AAV-IE vector combined with the CAG promoter efficiently transduced cochlear cells, with nearly 100% of hair cells and 80%-95% of supporting cells being transduced.
  2. Identification and Validation of Enhancers: Two CNEs within the SLC26A5 gene locus (SLC26A5-e1 and e2) collaboratively regulated the expression of SLC26A5.
  3. Functional Validation of Enhancers: SLC26A5-e1 knockout led to a progressive reduction in prestin expression during maturation, while SLC26A5-e1 + e2 double knockout completely abolished prestin expression, resulting in severe deafness.
  4. Engineering and Optimization of Enhancers: Synthetic enhancers combining e1p3 and e2p2 or e2p3 modules (such as b8) significantly improved gene expression efficiency and successfully restored hearing function in SLC26A5 knockout mice.

Conclusion

This study successfully identified and engineered enhancers of the SLC26A5 gene using the ARBITER workflow, achieving efficient and targeted gene therapy that restored hearing function in SLC26A5 knockout mice. This research not only deepens our understanding of the fundamental principles of enhancer-mediated gene expression but also provides new strategies and tools for the gene therapy of hereditary deafness.

Research Highlights

  1. Innovative Methodology: The ARBITER workflow provides a new method for the rapid and reliable dissection of enhancers of hearing loss genes.
  2. Efficient Enhancer Engineering: By optimizing enhancer modules, researchers successfully developed the highly efficient and specific enhancer b8, significantly improving gene expression efficiency.
  3. Therapeutic Potential: B8 enhancer-mediated gene therapy successfully restored hearing function in SLC26A5 knockout mice, demonstrating its potential application in the treatment of hereditary deafness.

Additional Valuable Information

The study also evaluated the therapeutic effect of the b8 enhancer in adult mice, showing that b8 could specifically transduce outer hair cells (OHCs) without causing hearing impairment. This finding provides further support for the clinical application of the b8 enhancer.

Through this research, we have not only deepened our understanding of the fundamental principles of enhancer-mediated gene expression but also provided new strategies and tools for the gene therapy of hereditary deafness.