Kesser B W, Hashisaki G T, Fletcher K, Eppard H, Holt J R
Department of Otolaryngology - Head and Neck Surgery, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Gene Ther. 2007 Aug;14(15):1121-31. doi: 10.1038/sj.gt.3302980. Epub 2007 Jun 14.
The confined fluid-filled labyrinth of the human inner ear presents an opportunity for introduction of gene therapy reagents designed to treat hearing and balance dysfunction. Here we present a novel model system derived from the sensory epithelia of human vestibular organs and show that the tissue can survive up to 5 days in vitro. We generated organotypic cultures from 26 human sensory epithelia excised at the time of labyrinthectomy for intractable Meniere's disease or vestibular schwannoma. We applied multiply deleted adenoviral vectors at titers between 10(5) and 10(8) viral particles/ml directly to the cultures for 4-24 h and examined the tissue 12-96 h post-transfection. We noted robust expression of the exogenous transgene, green fluorescent protein (GFP), in hair cells and supporting cells suggesting both were targets of adenoviral transfection. We also transfected cultures with a vector that carried the genes for GFP and KCNQ4, a potassium channel subunit that causes dominant-progressive hearing loss when mutated. We noted a positive correlation between GFP fluorescence and KCNQ4 immunolocalization. We conclude that our in vitro model system presents a novel and effective experimental paradigm for evaluation of gene therapy reagents designed to restore cellular function in patients who suffer from inner ear disorders.
人类内耳充满液体的封闭迷路为引入旨在治疗听力和平衡功能障碍的基因治疗试剂提供了机会。在此，我们展示了一种源自人类前庭器官感觉上皮的新型模型系统，并表明该组织在体外可存活长达5天。我们从因难治性梅尼埃病或前庭神经鞘瘤而在迷路切除时切除的26个人类感觉上皮中生成了器官型培养物。我们将滴度在10(5)至10(8)病毒颗粒/毫升之间的多重缺失腺病毒载体直接应用于培养物4至24小时，并在转染后12至96小时检查组织。我们注意到在毛细胞和支持细胞中外源转基因绿色荧光蛋白（GFP）有强烈表达，这表明两者都是腺病毒转染的靶标。我们还用携带GFP和KCNQ4基因的载体转染培养物，KCNQ4是一种钾通道亚基，突变时会导致显性进行性听力丧失。我们注意到GFP荧光与KCNQ4免疫定位之间存在正相关。我们得出结论，我们的体外模型系统为评估旨在恢复内耳疾病患者细胞功能的基因治疗试剂提供了一种新颖且有效的实验范式。
