Deeper knowledge about the role of the tumour microenvironment in cancer development and progression has resulted in new strategies such as gene-based cancer immunotherapy. DNA-based vaccines are intended to be expressed in antigen-presenting cells (e.g., dendritic cells, DCs), inducing anti-tumour responses of the adaptive immune system. Besides effective delivery systems (e.g., polyplexes) and the use of appropriate adjuvants, an optimized cargo is important for successful DNA vaccines. In this work, the concept of DC-focused transcriptional targeting was tested by using a plasmid encoding for the luciferase reporter gene under the control of a derivative of the fascin1 gene promoter (pFscnLuc), comprising an upstream DC-specific enhancer region and the proximal core promoter. DC-focused activity of this reporter construct was confirmed in vitro, where according polyplexes led to higher RLU values (up to 6-fold) in the DC-like cell line DC2.4 compared to polyplexes formed with the standard plasmid pCMVLuc encoding for luciferase under the control of the strong ubiquitously active cytomegalovirus promoter and enhancer. The two plasmids were also compared in vivo by intravenous administration. Linear polyethylenimine 22kDa (LPEI) as well as succinylated branched PEI 25kDa (succPEI, 10% succinylation) were used as effective pDNA carriers. In contrast to succPEI, LPEI showed a strong preference for mediating gene transfer to the lungs. The organ-specific expression profile of pFscnLuc versus pCMVLuc was promising for both carriers with favourable activity in the spleen as a central immune organ. Compared to pCMVLuc, pFscnLuc/LPEI complexes exerted 10-fold lower lung expression, and pFscnLuc/succPEI complexes 12-fold higher spleen expression.