A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice
A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice
复制标题
DOI：
10.15252/emmm.201809396
复制
发表时间：
2019-01-01
影响因子：
11.1
通讯作者：
Reisinger, Ellen
中科院分区：
文献类型：
--
作者：
Al-Moyed, Hanan;Cepeda, Andreia P.;Reisinger, Ellen
传入听觉内毛细胞（IHC）突触的正常听觉和突触传递需要otoferlin。由编码otoferlin的OTOF基因突变引起的耳聋DFNB 9可以通过将野生型otoferlin cDNA转移到IHC中来治疗，这是困难的，因为这种转基因的尺寸很大。在这项研究中，我们产生了两个腺相关病毒（AAV），每个都含有一半的otoferlin cDNA。将这些双AAV 2/6半载体共注射到6至7日龄的otoferlin敲除（Otof（-/-））小鼠的耳蜗中导致全长otoferlin在高达50%的IHC中表达。在耳蜗中，otoferlin选择性地表达于听毛细胞。Otof（-/-）IHC的双AAV转导完全恢复快速胞吐作用，而耳铁蛋白依赖性囊泡补充达到野生型水平的35-50%。这些IHC中40%的突触带的损失无法避免，表明otoferlin在早期突触成熟中的作用。声短声诱发听觉脑干反应，阈值为40-60 dB。因此，我们提出，由双AAV载体介导的基因递送可能适合于治疗由大基因如OTOF突变引起的耳聋形式。
Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the OTOF gene encoding otoferlin, might be treated by transferring wild-type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno-associated viruses (AAVs), each containing half of the otoferlin cDNA. Co-injecting these dual-AAV2/6 half-vectors into the cochleae of 6- to 7-day-old otoferlin knock-out (Otof(-/-)) mice led to the expression of full-length otoferlin in up to 50% of IHCs. In the cochlea, otoferlin was selectively expressed in auditory hair cells. Dual-AAV transduction of Otof(-/-) IHCs fully restored fast exocytosis, while otoferlin-dependent vesicle replenishment reached 35-50% of wild-type levels. The loss of 40% of synaptic ribbons in these IHCs could not be prevented, indicating a role of otoferlin in early synapse maturation. Acoustic clicks evoked auditory brainstem responses with thresholds of 40-60 dB. Therefore, we propose that gene delivery mediated by dual-AAV vectors might be suitable to treat deafness forms caused by mutations in large genes such as OTOF.