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摘要: 目的 通过分析Otof-/-成年鼠在基因治疗前后各频率畸变产物耳声发射(distortion product otoacoustic emissions, DPOAE)和听觉脑干反应(auditory brainstem response, ABR)的阈值变化及其相关性, 探索治疗后听力恢复的评价方法。方法 经圆窗膜(round window membrane, RWM)路径, 向4周龄Otof-/-成年鼠内耳注射双AAV载体携带的内含肽介导的OTOF基因治疗体系, 检测治疗前后DPOAE与ABR阈值, 免疫荧光染色评估耳畸蛋白(otoferlin)恢复表达的内耳毛细胞比例及突触数量, 并进行统计学分析。结果 AAV-PHP.eB对耳蜗内毛细胞具有高转染率; 治疗体系纠正了Otof-/-成年鼠的听力, 且不影响野生型小鼠听功能。基因治疗后DPOAE阈值与ABR阈值变化在16 kHz处存在显著相关; 术后DPOAE略微上升, 但在术后2个月时出现恢复趋势。结论 基因治疗可显著恢复Otof-/-成年鼠听力, 基因治疗体系手术给药可能会引起听力损伤, 这需要更加精细、轻柔的操作, 以最大程度发挥基因治疗的作用。Abstract: Objective This study aims to analyze the threshold changes in distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) in adult Otof-/- mice before and after gene therapy,evaluating its effectiveness and exploring methods for assessing hearing recovery post-treatment.Methods At the age of 4 weeks,adult Otof-/- mice received an inner ear injection of a therapeutic agent containing intein-mediated recombination of the OTOF gene,delivered via dual AAV vectors through the round window membrane (RWM).Immunofluorescence staining assessed the proportion of inner ear hair cells with restored otoferlin expression and the number of synapses.Statistical analysis was performed to compare the DPOAE and ABR thresholds before and after the treatment.Results AAV-PHP.eB demonstrates high transduction efficiency in inner ear hair cells.The therapeutic regimen corrected hearing loss in adult Otof-/- mice without impacting auditory function in wild-type mice.The changes in DPOAE and ABR thresholds after gene therapy are significantly correlated at 16 kHz.Post-treatment,a slight increase in DPOAE was observeds,followed by a recovery trend at 2 months post-treatment.Conclusion Gene therapy significantly restored hearing in adult Otof-/- mice,though the surgical delivery may cause transient hearing damage.Precise and gentle surgical techniques are essential to maximize gene therapy's efficacy.
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