Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9—using a single overloaded AAV vector—is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

听力障碍是人类最常见的感觉障碍。到目前为止，重度聋受试者的康复依赖于通过人工耳蜗对听神经的直接刺激。但是，在某些形式的遗传性听力障碍中，Corti的器官在结构上是完整的，并且突变基因的治疗性替代可潜在地恢复自然听力。如果是otoferlin基因的缺陷（场外交易），这样的基因治疗会受到编码序列大小（〜6 kb）超出首选病毒载体腺相关病毒（AAV）的载运量（<5 kb）的阻碍。最近，双重AAV方法被用于部分恢复聋耳铁蛋白敲除的听力（Otof-KO）小鼠。在这里，我们雇用体外 和 体内 种方法来评估天然超载和全长合成的合成AAV的基因治疗潜力 奥托夫编码序列。产后早期注射入耳蜗Otof-免疫荧光标记和聚合酶链反应表明，KO小鼠超负荷的AAV驱动otoferlin在所有IHC中的约30％特异性表达。记录听觉脑干反应和行为分析表明听力部分恢复。总之，我们的结果表明，使用单个超载AAV载体对DFNB9进行病毒基因治疗确实是可行的，与双重AAV方法相比，降低了基因转移的复杂性。