PDF(Pubmed)
Abstract：
Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5\' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3\' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5\' to 3\' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.
摘要：
已使用基于双重腺相关病毒（AAV）的方法在临床前解决了耳聋引起的耳聋缺陷（OTOF）。然而,转导的时机，mRNA的重组，和用双杂交AAV方法的蛋白质表达方法以前没有被表征。这里,我们已经建立了一种离体测定法来确定双AAV介导的OTOF在小鼠胞囊毛细胞中表达的动力学。我们利用了两种不同的重组载体，它们包含DB-OTO，一个包含在毛细胞特异性Myo15启动子控制下的OTOF的5'部分，另一个是OTOF的3'部分。我们探索了Myo15启动子在小鼠胞囊毛细胞中的特异性，在OTOF缺陷小鼠模型中建立的DB-OTO离体剂量反应特征，并证明了腺毛细胞中AAV1的耐受性。此外，我们确定了与5'至3'向量的一对一比率的偏差，对重组OTOF的影响很小。最后,我们在体外14至21天建立了重组OTOFmRNA和蛋白质表达量的平台，恢复时间与体内模型相当。这些发现证明了离体模型系统用于探索表达动力学并建立双重AAV介导的OTOF表达的体内和离体恢复时机的实用性。
