Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia
Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia
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DOI：
10.1074/jbc.m604099200
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发表时间：
2006-10-06
影响因子：
4.8
通讯作者：
Engelhardt, John F.
中科院分区：
文献类型：
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作者：
Yan, Ziying;Lei-Butters, Diana C. M.;Engelhardt, John F.
用于临床应用的腺相关病毒血清型的选择受用于评价各种血清型的动物模型和模型系统的影响。在本研究中，我们试图比较rAAV 2/ 1，rAAV 2/ 2和rAAV 2/ 5转导在极化的人气道上皮细胞中使用新开发的普通柱层析方法纯化的病毒的生物学特性。结果表明，rAAV 2/ 1对人气道上皮细胞的顶端转导效率比rAAV 2/ 2和rAAV 2/ 5高100倍。人气道上皮细胞中的这种转导特征（rAAV 2/ 1 >> rAAV 2/ 2 = rAAV 2/ 5）与将这些载体经鼻施用至小鼠肺后观察到的特征（rAAV 2/ 5 > rAAV 2/ 1 >> rAAV 2/ 2）显著不同，强调了这两个物种之间这些血清型的转导差异。与rAAV 2/ 2和rAAV 2/ 5形成鲜明对比的是，rAAV 2/ 1以相似的效率转导人气道上皮的顶膜和基底外侧膜。然而，跨血清型转导的总体水平与载体内化无关。我们假设这些血清型的进入后处理的差异可能会影响顶端转导的效率。为此，我们测试了蛋白酶体抑制剂对增强三种血清型的核转位和基因表达的有效性。人极化气道上皮的rAAV 2/ 1顶端转导的增强比rAAV 2/ 2和rAAV 2/ 5低10倍。细胞分级分离研究表明，蛋白酶体抑制剂比rAAV 2/ 1更显著地增强rAAV 2/ 2和rAAV 2/ 5向细胞核的转运。这些结果表明，AAV 1在人气道上皮细胞中的转导生物学不同于AAV 2和AAV 5，这是由于改变了影响核转位的泛素/蛋白酶体敏感性。
The choice of adeno- associated virus serotypes for clinical applications is influenced by the animal model and model system used to evaluate various serotypes. In the present study, we sought to compare the biologic properties of rAAV2/ 1, rAAV2/ 2, and rAAV2/ 5 transduction in polarized human airway epithelia using viruses purified by a newly developed common column chromatography method. Results demonstrated that apical transduction of human airway epithelia with rAAV2/ 1 was 100- fold more efficient than rAAV2/ 2 and rAAV2/ 5. This transduction profile in human airway epithelia ( rAAV2/ 1 >> rAAV2/ 2 = rAAV2/ 5) was significantly different from that seen following nasal administration of these vectors to mouse lung ( rAAV2/ 5 > rAAV2/ 1 >> rAAV2/ 2), emphasizing differences in transduction of these serotypes between these two species. In stark contrast to rAAV2/ 2 and rAAV2/ 5, rAAV2/ 1 transduced both the apical and basolateral membraneofhumanairway epithelia with similar efficiency. However, the overall level of transduction across serotypes did not correlate with vector internalization. We hypothesized that differences in post-entry processing of these serotypes might influence the efficiency of apical transduction. To this end, we tested the effectiveness of proteasome inhibitors to augment nuclear translocation and gene expression from the three serotypes. Augmentation of rAAV2/ 1 apical transduction of human polarized airway epithelia was 10- fold lower than that for rAAV2/ 2 and rAAV2/ 5. Cellular fractionation studies demonstrated that proteasome inhibitors more significantly enhanced rAAV2/ 2 and rAAV2/ 5 translocation to the nucleus than rAAV2/ 1. These results demonstrate that AAV1 transduction biology in human airway epithelia differs from that ofAAV2andAAV5by virtue of altered ubiquitin/ proteasome sensitivities that influence nuclear translocation.