Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea.
Biomaterials. 2010 Mar;31(7):1858-64. doi: 10.1016/j.biomaterials.2009.11.019. Epub 2009 Dec 1.
Due to their intracellular permeability, protein transduction domains (PTDs) have been widely used to deliver proteins and peptides to mammalian cells. However, their performance in gene delivery has been relatively poor. To improve the efficiency of PTD-mediated gene delivery, we synthesized a new peptide, KALA-Antp (K-Antp), which contains the sequences for PTD of the third alpha-helix of Antennapedia (Antp) homeodomain and the fusogenic peptide KALA. In this configuration, Antp is designed to provide the cell permeation capacity and nuclear localization signal, while the KALA moiety to promote cellular entry of the peptide-DNA complex. An optimal K-Antp/DNA formula was nearly 400-600 fold more efficient than Antp or poly-lysine-Antp (L-Antp) in gene delivery, and comparable or superior to a commercial liposome. The K-Antp-mediated plasmid DNA transfection not only exhibited temperature sensitivity, reflecting the involvement of an endocytosis-mediated gene transfer mechanism similar to other known PTDs, but also temperature insensitivity, suggesting the role of an energy-independent mechanism. Incorporation of an endosomolytic polymer polyethylenimine (PEI) into the system or treatment with chloroquine further increased the efficiency of K-Antp-mediated gene delivery. These results demonstrate the potential of the combinatorial use of KALA, Antp and PEI in the development of efficient PTD-derived gene carriers.
由于其细胞内通透性，蛋白转导结构域（PTDs）已被广泛用于将蛋白质和肽递送到哺乳动物细胞。然而，它们在基因传递中的性能相对较差。为了提高 PTD 介导的基因传递效率，我们合成了一种新的肽，KALA-Antp（K-Antp），它包含 Antennapedia（Antp）同源域第三α螺旋的 PTD 序列和融合肽 KALA。在这种构象中，Antp 被设计提供细胞渗透能力和核定位信号，而 KALA 部分则促进肽-DNA 复合物进入细胞。最佳的 K-Antp/DNA 配方在基因传递中的效率比 Antp 或聚赖氨酸-Antp（L-Antp）高近 400-600 倍，与商业脂质体相当或更优。K-Antp 介导的质粒 DNA 转染不仅表现出温度敏感性，反映了一种内吞介导的基因转移机制的参与，类似于其他已知的 PTDs，而且还表现出温度不敏感性，表明存在一种能量非依赖性机制的作用。将内溶酶体聚合物聚乙烯亚胺（PEI）纳入该系统或用氯喹处理进一步提高了 K-Antp 介导的基因传递效率。这些结果表明，KALA、Antp 和 PEI 的组合使用在开发有效的 PTD 衍生基因载体方面具有潜力。
