Comparative analysis of RNA versus protein splicing in dual AAV-mediated gene therapy in a mouse model of DFNB9 deafness

Mengzhao Xun; Xintai Fan; Hui Wang; Jingjing Zhao; Gongrui Tang; Weida Zhang; Shaowei Hu; Longlong Zhang; Daqi Wang; Yuxin Chen; Jianping Liu; Honghai Tang; Geng-Lin Li; Bing Chen; Yilai Shu

doi:10.1016/j.ymthe.2025.10.002

Comparative analysis of RNA versus protein splicing in dual AAV-mediated gene therapy in a mouse model of DFNB9 deafness

Mol Ther. 2026 Jan 7;34(1):203-215. doi: 10.1016/j.ymthe.2025.10.002. Epub 2025 Oct 6.

Authors

Mengzhao Xun¹, Xintai Fan¹, Hui Wang¹, Jingjing Zhao¹, Gongrui Tang², Weida Zhang¹, Shaowei Hu¹, Longlong Zhang¹, Daqi Wang¹, Yuxin Chen¹, Jianping Liu³, Honghai Tang⁴, Geng-Lin Li⁵, Bing Chen⁶, Yilai Shu⁷

Affiliations

¹ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China; NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai 200031, China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Fudan University, Shanghai 200031, China.
² The First Affiliated Hospital of Harbin Medical University, Harbin 150000, China.
³ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China; NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai 200031, China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Fudan University, Shanghai 200031, China. Electronic address: doctorentliu123@sina.com.
⁴ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China; NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai 200031, China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Fudan University, Shanghai 200031, China. Electronic address: hhtang16@fudan.edu.cn.
⁵ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China; NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai 200031, China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Fudan University, Shanghai 200031, China. Electronic address: genglin.li@fdeent.org.
⁶ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China; NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai 200031, China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Fudan University, Shanghai 200031, China. Electronic address: bingchen@fudan.edu.cn.
⁷ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China; NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai 200031, China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Fudan University, Shanghai 200031, China. Electronic address: yilai_shu@fudan.edu.cn.

PMID: 41058172
PMCID: PMC12925799 (available on 2027-01-07)
DOI: 10.1016/j.ymthe.2025.10.002

Abstract

Pathological mutations in the OTOF gene cause autosomal recessive deafness 9 (DFNB9). Although dual-adeno-associated virus (AAV) gene replacement therapy has been shown to partially rescue the hearing of patients with DFNB9, the therapeutic effects still need further exploration. To investigate the impact of different recombination strategies on the efficacy of OTOF gene therapy, we constructed five dual-AAV1 therapeutic agents using RNA or protein splicing principles. Based on the recombination strategy using RNA splicing, the AAV1-AK system rescued the hearing of Otof^-/- mice to 55-70 dB, which outperformed the AAV1-AP and AAV1-TS systems. Based on protein splicing, the AAV1-intein system rescued the hearing of Otof^-/- mice to 35-70 dB, which outperformed that of AAV1-AK, and the effects lasted up to 12 months after injection in both newborn and adult mice. The efficacy of otoferlin re-expression and the number and functional restoration of ribbon synapses in the AAV1-intein system were also better than the AAV1-AK system. These results show that protein recombination is more efficient than nucleic acid recombination for gene therapy in DFNB9. Therefore, this work provides not only data for optimization of DFNB9 gene therapy but also a reference for the delivery of other large genes.

Keywords: DFNB9; RNA splicing; adeno-associated virus; dual AAV; gene therapy; protein splicing.

MeSH terms

Animals
Deafness* / genetics
Deafness* / therapy
Dependovirus* / genetics
Disease Models, Animal
Genetic Therapy* / methods
Genetic Vectors* / administration & dosage
Genetic Vectors* / genetics
Humans
Membrane Proteins* / genetics
Mice
Mice, Knockout
Protein Splicing*
RNA Splicing*

Substances

Membrane Proteins