593. Anti-Angiogenic Therapy for Pancreatic Cancer by Systemic Delivery of Messenger RNA Using Polyplex Nano Micelle

Abstract

In pancreatic cancer, the access of therapeutic reagents to cancer cells was restricted by thick fibrotic tissue, and thus anti-angiogenic therapy targeting endothelial cells, which are more accessible, is a promising strategy. Gene therapy based on messenger RNA (mRNA) delivery allows for sustainable introduction of anti-angiogenic factors without the risks associated with DNA delivery, such as insertion mutagenesis, and uncontrollably prolonged transgene expression. Systemic intravenous injection is a proper delivery route for mRNA to access endothelial cells from blood vessels with a simple injection procedure. Because mRNA is highly susceptible to enzymatic degradation in the blood circulation, mRNA carrier with high stability is required. In this study, we used polyplex nanomicelles, prepared from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymers (Adv Drug Deliv Rev 2001, 47, 113-131), and a cholesterol moiety (Chol) was introduced to the block copolymer to stabilize the nanomicelles by hydrophobic interaction. In the cationic segment of the block copolymer, a polyaspartamide with four aminoethylene repeating units in the side chain (PAsp(TEP)) was used, which has high endosomal escaping capability and mRNA stabilizing effect (J Am Chem Soc 2014, 136, 12396-12405). In quantitative PCR (qPCR) analysis of nuclease resistance after in vitro incubation in 50% serum, Chol introduction failed to increase mRNA stability. In contrast, after intravenous injection to mice, the nanomicelles with Chol showed significantly enhanced mRNA retention in the blood circulation in qPCR analysis, when compared to those without Chol. These results indicate that the stabilizing effect of Chol is obvious especially in the harsh environment of blood, where nanomicelles are exposed to large amount of polyanion, such as proteoglycans on the cell surface. Indeed, nanomicelles with Chol showed enhanced resistance to dissociation by dextran sulfate compared to those without Chol. Eventually, when luciferase mRNA was introduced to the mice subcutaneously inoculated with human pancreatic adenocarcinoma (BxPC3), the nanomicelles with Chol showed enhanced luciferase expression in the tumor tissue, compared to those without Chol. Then, anti-angiogenic treatment was performed to this BxPC3 model, using mRNA encoding sFlt-1, a soluble form of vascular endothelial growth factor (VEGF) receptor, which inhibits VEGF signaling by entrapping VEGF protein. In the evaluation of tumor volume, the nanomicelles with Chol exhibited significant growth inhibitory effect, whereas those without Chol failed to show detectable effect. In immunohistochemical staining of vascular endothelial cells, the nanomicelles with Chol induced significant reduction of vascular density in the tumor tissue, indicating that the anti-angiogenic effect contributed to the inhibition of tumor growth after injection the nanomicelles with Chol. In conclusion, we succeeded in anti-angiogenic treatment of intractable pancreatic cancer by systemic mRNA delivery using stabilized nanomicelles. This mRNA delivery system can also be applied to the treatment of various diseases in the future.