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

质粒DNA （pDNA）已成为一类重要的大分子药物，适用于非病毒基因治疗和DNA疫苗接种。在体内应用pDNA被认为比病毒更安全，因为可能产生的不良影响更小。然而，越来越多的证据表明，细菌而不是哺乳动物的DNA可以激活免疫能力细胞，特别是巨噬细胞，这引起了人们的关注。然而，巨噬细胞对pDNA的细胞摄取机制尚不完全清楚。为了阐明pDNA的作用机制，我们利用体外培养的表达A类清除率受体（SRA）的中国仓鼠卵巢细胞和SRA敲除小鼠的腹腔巨噬细胞，研究了pDNA的结合和摄取。我们发现pDNA在小鼠腹膜巨噬细胞中的结合和摄取是通过一种特定的机制介导的，这种机制基于聚阴离子的三维结构。此外，我们还研究了含有未甲基化CpG基序的pDNA与阳离子脂质体复合物诱导的细胞因子分泌。巨噬细胞在该复合物刺激下产生大量肿瘤坏死因子-α (TNF-α)。然而甲基化的pDNA和小牛胸腺DNA与阳离子脂质体复合物可诱导TNF-α和IL-6的产生，表明这些反应不依赖于CpG基序。这些结果表明，当pDNA与脂质体制剂结合时，pDNA通过不依赖CpG基序的方式激活体外培养的巨噬细胞。这些发现将为基因治疗和DNA疫苗接种中pDNA递送的优化提供重要依据。

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.