Novel gene delivery systems using synthetic oligopeptide and MAP

我们描述了一种由合成肽介导的基因转移新方法。这种方法是基于利用树突肽发展成多抗原肽（MAP）。将四聚支链肽和八聚支链肽作为转染试剂，与赖氨酸残基组成的单体肽、聚赖氨酸进行了比较。单肽的转染活性基本不高，接近背景水平，但在树突状肽中，尤其是八聚体肽的转染效率较高。采用3、6、9、12、15聚八聚体多肽研究了支链长度对赖氨酸残基的影响。长度在6聚的短肽是不有效的，但12或15聚的肽是更有效的DNA转染。在6个培养的哺乳动物细胞HeLaS3、NIH-3T3、L929、cv - 1、COS-7和HepG2中检测了树突状肽对DNA转染的能力。多肽产生了一个有效的基因转移与各种细胞系。这些结果表明，阳离子肽的支链结构更有利于基因的有效传递。利用F-moc化学合成了由9个氨基酸残基组成的不同序列的寡肽。它们含有赖氨酸、亮氨酸、色氨酸、半胱氨酸或丝氨酸。根据pSV2cat质粒DNA向细胞的功能转移来检测这些合成的低聚肽的转染能力。由赖氨酸、色氨酸和半胱氨酸组成的特异性寡肽具有促进基因向哺乳动物细胞转移的能力。KLKLCLKLK二聚体有部分赖氨酸被亮氨酸取代的表达。将部分或全部亮氨酸替换为色氨酸更能促进基因传递，特别是KWKWCWKWK二聚体的转染效率最高。这些结果表明疏水性氨基酸和阳离子氨基酸都是寡肽传递基因所必需的，色氨酸比亮氨酸更有利。此外，值得注意的是，具有N端或c端半胱氨酸残基并形成线性二聚体的CWKWKWKWK和KWKWKWKWC根本不介导基因传递。这表明由S-S键形成所产生的二聚体肽的结构是决定是否将DNA传递到细胞的关键。这表明KWKWCWKWK二聚体中的Trp对彼此之间特别接近，提供了更强的偶对。KWKWCWKWK二聚体的CD光谱反映了它们独特的结构。少

We describe a novel approach for gene transfer mediated by synthetic peptides. This method was base on use of dendritic peptides that developed as the multiple antigen peptides(MAP). The tetra and octameric branched peptide as transfection reagent were compared with the monomeric peptide consisted of lysine residues, polylysine. The monomeric peptide was essentially little activity of transfection, near the background level, but in the dendritic peptides, especially, octameric peptides showed higher transfection efficiency. Effects of chain length of branched lysine residue were studied using 3, 6, 9, 12, 15 mer octameric peptides. Short length peptides up to 6 mer were not efficient, but 12 or 15 mer peptides were more efficient for DNA transfection. The ability of the dendritic peptides on DNA transfection was examined in six cultured mammalian cells : HeLaS3, NIH-3T3, L929, CV-l, COS-7 and HepG2. The peptides yielded an efficient gene transfer with a variety of cell lines. These stu … More dies suggested that branched structure in the cationic peptide was more favorable for efficient gene delivery.Oligopeptide of various sequences constituted by 9 amino acids residue were synthesized by F-moc chemistry. They contain lysine, leucine, tryptophan, cysteine or serine. The transfection ability of these synthetic oligo-peptides were examined in terms of the functional transfer of pSV2cat plasmid DNA into cells. Specific oligopeptide composed of lysine, tryptophan and cystein have ability (facilitate) of gene transfer to mammalian cells. Dimer of KLKLCLKLK substituted a part of lysine by leucine showed somewhat expression. A substitution a part or all of leusine to tryptophan still more promote gene delivery, especially dimer of KWKWCWKWK showed highest efficient transfection. These results indicated hydrophobic amino acid as well as cationic amino acid is necessary for gene delivery by oligopeptide and tryptophan is more favorable than leucine. Furthermore, it is worthy of notice that CWKWKWKWK and KWKWKWKWC which possess N or C-terminal cysteine residue and form linear dimer not at all mediate gene delivery. It indicates that the structure of dimer peptide produced by formation of S-S bond is critical in deciding whether to deliver DNA to cells. It suggests that Trp pairs in KWKWCWKWK dimer is in particular close each other and provides the stronger couplet. The CD spectra of KWKWCWKWK dimer reflected their distinctive structure. Less