Cochlear expression of a dominant-negative GJB2R75W construct delivered through the round window membrane in mice
Cochlear expression of a dominant-negative GJB2R75W construct delivered through the round window membrane in mice
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DOI：
10.1016/j.neures.2007.03.006
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发表时间：
2007-07-01
影响因子：
2.9
通讯作者：
Smith, Richard J. H.
中科院分区：
文献类型：
--
作者：
Maeda, Yukihide;Fukushima, Kunihiro;Smith, Richard J. H.
开发内耳基因传递方法是研究听力功能和基因治疗的重要步骤。将脂质体或腺病毒载体应用于完整的圆窗膜（RWM）提供了非创伤外源基因转移的可能性。GJB2编码缝隙连接蛋白Connexin26，该蛋白在内耳钾循环中起关键作用。GJB的R75W等位基因是一个很好的突变，它通过显性负作用机制在DFNA3上引起耳聋。本研究将质粒pGJB2(R75W)- egfp与N-[1-（2,3-二油基氧基）丙基]N，N，N-三甲基甲基硫酸铵：胆固醇进行脂质体复配，并应用于小鼠RWM。在治疗后3天（3 d），免疫组化显示GJB2(R75W)-eGFP基因在耳蜗内、外柱细胞、外毛细胞、克劳迪氏细胞以及螺旋缘和韧带中表达。治疗后1、2、3 d通过听觉脑干反应测试发现听力明显下降；治疗后5 d，听力水平恢复到对照水平。这些数据证实了GJB2（R75W）通过显性负作用诱导成熟耳蜗功能损伤，重要的是外源基因RWM应用是测试其对听力影响的可行方法。(C) 2007爱思唯尔爱尔兰有限公司和日本神经科学学会。版权所有。
Development of a gene-delivery method to the inner ear is an essential step for studies of hearing function and gene therapy. Application of liposomes or adenoviral vectors onto the intact round window membrane (RWM) offers the possibility of atraumatic exogenous gene transfer. GJB2 encodes the gap junction protein Connexin26, which plays a crucial role in potassium recycling in the inner ear. The R75W allele of GJB is a well-characterized mutation that causes deafness at the DFNA3 through a dominant-negative mechanism of action. In this study, a plasmid vector, pGJB2(R75W)-eGFP, was lipocomplexed with N-[1-(2,3-Dioleoloxy)propyl]N,N,N-trimethylammonium methylsulfate: cholesterol and applied onto mouse RWM. At 3 days (3 d) post-treatment, immunohistochemistry demonstrated GJB2(R75W)-eGFP transgene expression in the cochlea in: inner and outer pillar cells, outer hair cells, Claudius cells and, in the spiral limbus and ligament. Significant hearing loss was detected by auditory brainstem response testing after 1, 2 and 3 d post-treatment; hearing levels returned to control levels at 5 d post-treatment. These data confirm that GJB2(R75W) induces functional impairment in the mature cochlea through a dominant negative effect, and importantly, that RWM application of exogenous genes is a feasible method to test their impact on hearing. (C) 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.