Over the past several decades, there have been numerous attempts to utilize synthetic dsRNA to control tumor growth in animal models and clinical trials. Recently, it has become clear that intracellular dsRNA is more effective than extracellular dsRNA in promoting apoptosis and orchestrating adaptive immune response. To overcome the difficulty in delivering a large dose of synthetic dsRNA into tumors, while avoiding systemic toxicity we propose to deliver a RNA replicase-based plasmid DNA, hypothesizing that the dsRNA generated by the replicase-based plasmid in tumor cells will inhibit tumor growth. We evaluated the anti-tumor activity of a plasmid (pSIN-beta) that encodes the sindbis RNA replicase genes in mice with model tumors (TC-1 lung cancer cells or B16 melanoma cells) and compared it to a traditional pCMV-beta plasmid. In cell culture, transfection of tumor cells with pSIN-beta generated dsRNA. In mice with model tumors, pSIN-beta more effectively inhibited tumor growth than pCMV-beta, and in some cases, eradicated the tumors. RNA replicase-based plasmid may be exploited to generate intracellular dsRNA to control tumor growth. The feasibility of further improving the antitumor activity of the RNA replicase-based plasmid by targeting it into tumors cells was also evaluated. An epidermal growth factor (EGF)-conjugated, PEGylated cationic liposome was developed to deliver the RNA replicase-based plasmid, pSIN-beta, into EGFR-over-expressing human breast cancer cells (MDA-MB-468) in vitro and in vivo. Delivery of the pSIN-beta using the EGF receptor-targeted liposome more effectively controlled the growth of MDA-MB-468 tumors in mice than using un-targeted liposome. Finally the potential of further improving the antitumor activity of the pSIN-beta plasmid by incorporating interleukin-2 (IL2) gene into the plasmid was investigated. The resultant pSIN-IL2 plasmid was delivered to mouse melanoma cells that over-express the sigma receptor. The pSIN-IL2 plasmid was more effective at controlling the growth of B16 melanoma in mice when complexed with sigma receptor targeted AA-PEG-liposomes than with the untargeted liposomes. Importantly, the pSIN-IL2 plasmid was more effective than pSIN-beta plasmid at controlling the growth of B16 melanoma in mice, and B16-bearing mice that were treated with pSIN-IL2 had an elevated number of activated CD4+, CD8+, and natural killer cells, compared to those treated with pSIN-beta. / text