Tuyaerts Sandra, Michiels Annelies, Corthals Jurgen, Bonehill Aude, Heirman Carlo, de Greef Catherine, Noppe Sofie M, Thielemans Kris
Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the Vrije Universiteit Brussel (VUB), Laarbeeklaan 103/E, 1090 Brussels, Belgium.
Cancer Gene Ther. 2003 Sep;10(9):696-706. doi: 10.1038/sj.cgt.7700622.
Genetically modified dendritic cells (DC) constitute a promising approach in cancer immunotherapy. Viral gene delivery systems have been shown to be very efficient strategies, but safety concerns for their clinical use in immunotherapy remain an important issue. Recently, the technique of mRNA electroporation was described as a very efficient tool for the genetic modification of human monocyte-derived DC. Here, we show that transgene expression can be modulated by varying the amount of mRNA used for electroporation. We document that CD40 ligation leads to a significant production of IL-12 by the electroporated DC, although the level of IL-12 production is somewhat lower than for non- or mock-electroporated DC. Furthermore, we show that the electroporated DC can be frozen and thawed without loss of viability or function and that Influenza virus Matrix Protein 1 mRNA electroporated DC are capable of inducing a memory cytotoxic T lymphocyte response and are more potent in doing so than mRNA-pulsed DC. Similar results were obtained with MelanA/MART-1 mRNA electroporated DC. These results clearly indicate that mRNA-electroporated DC represent powerful candidates for use as tumor vaccines and could constitute an improvement compared with vaccines using peptide-pulsed DC.
基因修饰的树突状细胞（DC）是癌症免疫治疗中一种很有前景的方法。病毒基因递送系统已被证明是非常有效的策略，但它们在免疫治疗临床应用中的安全性问题仍然是一个重要问题。最近，mRNA电穿孔技术被描述为一种对人单核细胞衍生的DC进行基因修饰的非常有效的工具。在此，我们表明可以通过改变用于电穿孔的mRNA量来调节转基因表达。我们证明，尽管IL-12的产生水平略低于未电穿孔或模拟电穿孔的DC，但CD40连接可导致电穿孔的DC大量产生IL-12。此外，我们表明电穿孔的DC可以冷冻和解冻而不丧失活力或功能，并且流感病毒基质蛋白1 mRNA电穿孔的DC能够诱导记忆性细胞毒性T淋巴细胞反应，并且在这样做时比mRNA脉冲的DC更有效。用MelanA/MART-1 mRNA电穿孔的DC也获得了类似的结果。这些结果清楚地表明，mRNA电穿孔的DC是用作肿瘤疫苗的有力候选者，并且与使用肽脉冲DC的疫苗相比可能构成一种改进。
