Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation
The “modular promoter (MP)” system enables highly efficient and specific expression of therapeutic transgenes in mature DCs. (a) Schematic outline of the dual vector “modular promoter” system. Ad5E/P-510/mHSF1 drives expression of mHSF1 under the control of the cell type- and maturation-specific human CD83 promoter, which in turn induces specific induction of therapeutic transgenes MelanA and Il-12 by binding to the Hsp70B′−29/−242 HRE encoded by Ad5MP2. (b, c) XS52 cells were cotransfected with a total amount of 2 μg of plasmid vectors pP-510/mHSF1, pEs/P-510/mHSF1, or pEas/P-510/mHSF1 in combination with plasmid vectors encoding the HRE alone (“pHsp70B′−29/−242”) or the modular promoter system (“pMP2”). As a control, the empty pGL3-Basic vector (“Basic”) was used. (d, e) Human immature DCs were cotransduced with Ad5MP2 or Ad5MelA in combination with Ad5Luc1, Ad5mHSF1, Ad5P-510/mHSF1, Ad5Es/P-510/mHSF1, or Ad5Eas/P-510/mHSF1. Cells were either left immature or were matured by adding 0.1 ng/mL LPS. (b–e) Twenty hours after transfection, cell lysates were analyzed by Western Blot for expression of MelanA and beta-actin (b, d) and cell culture supernatants by ELISA for content of IL-12p40 and IL-12p70 (c, e). Either one representative (b, d) or data mean ± SEM from three independent experiments (c, e) is shown. ; ; bars without annotation are not significant (), one-way ANOVA (c) or two-way ANOVA (e) with Bonferroni’s Multiple Comparison post hoc test.
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