Development of peptide vectors containing Aid residues for gene therapy

TV-XIIa与10聚赖氨酸(Lys)偶联的多肽载体[Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-OH；Ac：Acyyl，U：α-氨基异丁酸]可将20聚体寡核苷酸送入细胞，而10聚赖氨酸不能将其送入细胞内。然后，TV-XIIa本身被认为是传输系统中的重要角色。为了获得细胞摄取TV-XIIa本身的直接证据，并研究20聚体寡核苷酸的递送原理，合成了带有TV-XIIa氨基酸序列的荧光素标记多肽，并将其应用于活细胞，以直接观察TV-XIIa在活细胞中的行为。此外，为了了解三个不寻常的AIB残基在序列中的重要性，我们用ALA取代了它们，并检测了AIB→Ala替代类似物在活细胞中的行为。结果表明，荧光素标记的TV-XIIa多肽可以转运到细胞内，ALA完全取代AIB可抑制细胞摄取。含有AIB的多肽的移位似乎涉及一个不依赖能量的过程。为了优化将10聚体赖氨酸作为ODN相互作用部分添加到TV-XIIa中，将TV-XIIa衍生多肽的C-末端部分缩短为8-、6-和4-MERS的赖氨酸。4-聚体和6-聚体的衍生物不能将ODN送入细胞，而8-聚体和10-聚体的衍生物显著促进ODN的摄取。这些结果表明，TV-XIIa的膜通透性和Lys残基的数量可能是将ODN传递到细胞内的重要转运体。其次，以疏水氨基酸AIB和碱性氨基酸Lys为原料合成了两亲24-残基螺旋多肽[Ac-(U-K)6-NH2和Ac-(U-K)8-NH2]。我们发现，通过螺旋多肽和ODN之间的电相互作用形成的络合物被带入细胞。

The TV-XIIa-derived peptide vector [Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-OH; Ac: acetyl, U: α-aminoisobutyric acid (Aib)], which is conjugated between TV-XIIa and a 10-mer of lysine (Lys), could deliver 20-mer oligonucleotides (ODN) into cells, and the 10-mer of Lys alone was not capable of delivering them. Then, TV-XIIa itself is consider to be an important role in the delivery system. To obtain the direct evidences for the cellular uptake of TV-XIIa itself and investigate the delivery principles of the 20-mer oligonucleotides, a fluorescein-labeled peptide with the TV-XIIa amino acid sequence was synthesized and applied to living cells to directly observe the behavior of TV-XIIa itself in living cells. Furthermore, to understand the importance of the three unusual Aib residues in the sequence, they were replaced with Ala, and the behavior of the Aib→Ala substitutional analog in living cells was also examined. The results indicated that the fluorescein-labeled TV-XIIa peptide can translocate into cells and the all replacement of Aib with Ala inhibits the cellular uptake. The translocation of the Aib-containing peptide seems to involve an energy-independent process. To optimize the 10-mer of lysine added as a ODN interaction part to TV-XIIa, the C-terminal moiety of the TV-XIIa-derived peptide vector was shortened to 8-, 6-, and 4-mers of Lys. The 4- and 6-mer derivatives cannot delivery ODNs into cells, on the other hand, the 8- and 10-mer derivatives significantly promote the uptake of ODNs. These results indicated that both the membrane permeability of TV-XIIa and the number of Lys residues might be important rolls to delivery the ODNs into cellsNext, amphiphatic 24-residual helix peptides [Ac-(U-U-U-K)6-NH2 and Ac-(U-U-K)8-NH2] were synthesized using a hydrophobic amino acid, Aib and a basic one, Lys. We found that the complexes, which were formed via electric-interaction between the helix peptides and ODNs, are taken up into cells.