Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus
Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus
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DOI：
10.1089/107999000312702
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发表时间：
2000-01-01
影响因子：
2.3
通讯作者：
Hermonat, PL
中科院分区：
文献类型：
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作者：
Liu, Y;Santin, AD;Hermonat, PL
抗原呈递树突状细胞（DC）的遗传操作为刺激免疫应答提供了希望，特别是用于抗癌和抗病毒方案。由于腺相关病毒（AAV）已经显示出作为用于转导多种造血细胞类型的基因递送载体的前景，我们已经研究了AAV遗传改变DC的能力。在该分析中，我们修改了粘附单核细胞的标准粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）处理以产生DC。在我们的方案中，首先用AAV/GM-CSF/Neo载体感染粘附的单核细胞，并延迟2天加入IL-4以允许单核细胞短暂增殖。通过G418选择、GM-CSF分泌、GM-CSF RNA表达（逆转录酶-聚合酶链反应扩增[RT-PCR]）和细胞增殖证明了AAV介导的GM-CSF和Neo基因转导至单核细胞/DC前体中。由AAV/GR-CSF/Neo病毒感染以及随后的IL-4和肿瘤坏死因子-α（TNF-α）处理产生的细胞显示出与成熟DC一致的多种经典标志物。最后，在分选的CD 83（+）DC中也证明了AAV载体的染色体整合。这些数据有力地表明，AAV载体将用于DC的遗传操作，并表明GM-CSF基因的转导能够完全取代在成熟DC的产生中对外源GM-CSF的需要。
The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC, In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GR-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83(+) DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.