| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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
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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
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