Systemic dual delivery of a tumor-inhibiting plasmid expressing p16 and shRaf

Efforts to target tumors in vivo with high-molecular-weight plasmids have been hindered by inefficient delivery. Moreover, while siRNA and mRNA have targets in the cytosol, plasmid-based therapy must reach the nucleus to express its products. Consequently, many groups have abandoned robust self-amplifying plasmid-based therapies in favor of siRNA or mRNA. An efficient delivery system for plasmids could transform breast cancer treatment and represent an important step toward successful "gene" therapy in the clinic. Although selecting a tumor suppressor gene replacement, antiangiogenic gene expression, or shRNA expressed by plasmids, along with their synergy with adjuvant therapy, is important, these strategies are limited without an effective delivery system to the tumor. This report investigates whether polymeric carriers for plasmid-based therapy inhibit the growth of MDA-MB-231 cells. These triple-negative breast cancer cells display an enhanced Ras/Raf-1/ERK (ERK) pathway and lack the wild-type tumor suppressor p16. Using several polymeric carriers, we found that the plasmid expressing p16 and shRaf effectively suppressed tumor cells in vitro. Notably, despite low amounts in the polyplex, the histidine-lysine (HK) peptide carrier showed increased gene expression in the tumor xenografts compared to normal tissues. In contrast to tumor xenografts, no toxicity in normal tissues was observed with the p16shRaf therapy. The HK plasmid-based polyplex markedly reduced tumor size by approximately 75% compared to untreated controls. The antitumor effect of the p16shRaf-containing polyplex was due to increased apoptosis and decreased mitosis in tumor xenograft cells. • -Targeting multiple pathways with p16 and shRaf plasmid polyplexes increased the antitumor efficacy. • -Despite the low content of the histidine-lysine (HK) carrier, the negatively charged polyplex was specific to tumors in vivo. • -No toxicity in the normal tissues of mice was observed with the HK polyplex. • -HK polyplexes markedly decreased the size of human breast cancer xenografts • -The reduced tumor size was based on decreased cell division and enhanced apoptosis.