遺伝子医薬品の体内動態および細胞取り込み機構の解明に基づくデリバリー戦略の確立

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Plasmid DNA (pDNA) has become an important class of macromolecular agent suitable for non-viral gene therapy as well as DNA vaccination. In vivo application of pDNA is thought to be safer than that of viruses because there is less potential for adverse effects. However, concerns have been raised since there is increasing evidence suggesting that bacterial, but not mammalian, DNA activates immune competent cells, especially macrophages. However, the cellular uptake mechanism of pDNA by macrophages is not yet fully understood. In order to elucidate the mechanism, the binding and uptake of pDNA were studied in vitro using cultured Chinese hamster ovary cells expressing the class A scavenger receptor (SRA) and peritoneal macrophages from SRA-knockout mice. We found that pDNA binding and uptake in mouse peritoneal macrophages are mediated by a specific mechanism to some defined polyanions based on three-dimtensional structure of polyanions. Furthermore, we investigated the cytokine secretion induced by pDNA containing unmethylated CpG motifs complexed with cationic liposomes. A significant amount of tumor necrosis factor-α (TNF-α) was produced from the macrophages upon stimulation with the complex. However methylated pDNA and calf thymus DNA complexed with the cationic liposomes could induce TNF-α and IL-6 production, indicating that these responses were not dependent on CpG motifs. These results suggest that pDNA becomes active to stimulate the cultured macrophages in vitro through CpG motif independent manner when it is combined with the liposome formulations. These findings would be an important basis for optimization of pDNA delivery in gene therapy and DNA vaccination.