Héma-Québec, Québec, QC, Canada.
J Gene Med. 2011 Sep;13(9):451-61. doi: 10.1002/jgm.1588.
Investigation of the molecular processes that control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte-associated pathogenesis. Adenovirus-mediated gene transfer provides a powerful tool for investigating these processes. However, we observed variation in transgene expression among normal human peripheral blood B lymphocytes from different donors and at distinct stages of differentiation. It is recognized that efficient gene transfer is highly dependent on the intracellular route by which the viruses travel within the host cell. Thus, we aimed to examine this aspect in the present study.
We analyzed the binding, uptake, intracellular trafficking and fate of CY3-labelled Ad5/F35 vectors in lymphoid cell lines and primary B cells. Furthermore, we decreased protein synthesis levels and rapid endocytosis in a plasma cell line exhibiting a high level of protein synthesis activity and activated transcription and endocytosis in primary B cells, which are less active than plasma cells.
Major differences in intracellular trafficking pattern between B cells and plasma cell line U266 were identified that explain the observed divergence in transgene expression efficiency. Importantly, modification of the transcriptional or translational activity of U266 cells reverted the Ad5/F35 endocytic trafficking to that seen in B cells, with a loss of transgene expression, whereas activation of B cells with phorbol 12-myristate 13-acetate had the opposite effects.
Taken together, these results suggest that Ad5/F35 is more efficiently transduced in cells with a strong transcriptional activity as a result of differences in intracellular trafficking. This finding extends our current knowledge of the mechanisms of adenovirus-mediated gene transfer.
研究控制淋巴细胞发育和功能的分子过程对于我们理解体液免疫以及与淋巴细胞相关的发病机制至关重要。腺病毒介导的基因转移为研究这些过程提供了强大的工具。然而，我们观察到来自不同供体和分化阶段的正常人外周血 B 淋巴细胞中转基因表达存在差异。人们认识到，有效的基因转移高度依赖于病毒在宿主细胞内的胞内途径。因此，我们旨在在本研究中检查这一方面。
我们分析了 CY3 标记的 Ad5/F35 载体在淋巴样细胞系和原代 B 细胞中的结合、摄取、细胞内转运和命运。此外，我们降低了浆细胞瘤系中蛋白合成水平和快速内吞作用，该浆细胞瘤系具有高水平的蛋白合成活性，并激活了原代 B 细胞中的转录和内吞作用，原代 B 细胞的活性低于浆细胞瘤系。
鉴定了 B 细胞和浆细胞瘤系 U266 之间细胞内转运模式的主要差异，这些差异解释了观察到的转导效率差异。重要的是，改变 U266 细胞的转录或翻译活性使 Ad5/F35 的内吞转运恢复为 B 细胞所见，导致转导基因表达丢失，而用佛波醇 12-肉豆蔻酸 13-乙酸酯激活 B 细胞则产生相反的效果。
总之，这些结果表明，由于细胞内转运的差异，具有强转录活性的细胞中 Ad5/F35 的转导效率更高。这一发现扩展了我们对腺病毒介导基因转移机制的现有认识。
