Hashida H, Miyamoto M, Cho Y, Hida Y, Kato K, Kurokawa T, Okushiba S, Kondo S, Dosaka-Akita H, Katoh H
Department of Surgical Oncology, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan.
Br J Cancer. 2004 Mar 22;90(6):1252-8. doi: 10.1038/sj.bjc.6601680.
Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.
有效的基因治疗依赖于将治疗性基因高效地转移到靶细胞。然而，目前的技术都无法满足基因治疗所需的所有要求，因为哺乳动物细胞的质膜和核膜对于使用合成递送系统进行基因转移而言是紧密的屏障。人类免疫缺陷病毒1型（HIV-1）Tat蛋白的蛋白转导结构域（PTD）通过膜去稳定化极大地促进了蛋白质转移。我们合成了含有Tat PTD（TAT-pK）或其他序列的聚赖氨酸肽，并研究了它们作为基因转移剂的潜力。合成的多肽TAT-pK保留了DNA结合功能，并介导报告基因递送至培养细胞。RGD基序与诱导细胞活化的α整合素以低亲和力结合。合成并测试了两种对照多肽GGG-pK和RGD-pK，但它们的基因转移能力比TAT-pK弱。在氯喹或氯化铵存在下，TAT-pK介导的基因转移增强，在大多数测试的癌细胞系中，其增强程度大于阳离子脂质介导的基因转移。这些数据表明，TAT-pK可能是一种有效的候选递送载体，可促进基因转移，这取决于内吞途径。我们得出结论，TAT-pK/DNA复合物作为包装治疗性基因并将其转导至哺乳动物细胞的最小单元是有用的。
