This study reports on the development of a novel lipid combination that permits the efficient and highly selective delivery of plasmid DNA (pDNA) to immune cells in the spleen. Using DODAP, an ionizable lipid that was previously thought to be inefficient for gene delivery, we show for the first time, that this ignored lipid can be successfully used for efficient and targeted gene delivery in vivo, but only when combined with DOPE, a specific helper lipid. Using certain DODAP and DOPE ratios resulted in the formation of lipid nanoparticles (LNPs) with a ~ 1000-fold higher gene expression, and this expression was specific for the spleen, making it the most spleen-selective system for transfection using pDNA. The developed DODAP/DOPE-LNPs target immune cells in the spleen via receptors for complement C3 and this pathway is critical for efficient gene expression. We hypothesize that the high spleen transfection activity of DODAP/DOPE-LNPs is caused by the promotion of gene expression associated with B cell activation via complement receptors. LNPs encapsulating tumor-antigen encoding pDNA showed both prophylactic and therapeutic anti-tumor effects. The optimized LNPs resulted in the production of different cytokines and antigen-specific antibodies as well as exerting antigen-specific cytotoxic effects. This study revives the use of DODAP in gene delivery and highlights the importance of using appropriate lipid combinations for delivering genes to specific cells. Display omitted •A novel lipid combination for efficient and selective delivery of plasmid DNA to immune cells in the spleen was developed.•The lipid system is composed of the pH-sensitive cationic lipid DODAP, in combination with the fusiogenic lipid DOPE.•The optimized DODAP-DOPE lipid nanoparticles (LNPs) achieve high and selective gene expression in the spleen.•DODAP-DOPE LNPs encapsulating tumor-antigen encoding pDNA showed both prophylactic and therapeutic anti-tumor effects.•The optimized LNPs resulted in the production of different cytokines and antibodies as well as exerting cytotoxic effects.