Development of peptide vectors containing Aid residues for gene therapy

TV-XIIa衍生的肽载体[Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-OH; Ac：乙酰基，U：α-氨基异丁酸（Aib）]，其缀合在TV-XIIa和10-mer赖氨酸（Lys）之间，可以将20-mer寡核苷酸（ODN）递送到细胞中，而单独的10-mer Lys不能递送它们。然后，TV-XIIa本身被认为是一个重要的作用，在交付系统。为了获得细胞摄取TV-XIIa本身的直接证据并研究20-mer寡核苷酸的递送原理，合成了具有TV-XIIa氨基酸序列的荧光素标记肽，并将其应用于活细胞以直接观察TV-XIIa本身在活细胞中的行为。此外，为了了解序列中三个不常见的Aib残基的重要性，将它们替换为Ala，还检查了Aib→Ala取代类似物在活细胞中的行为。结果表明，荧光素标记的TV-XIIa肽可以转位到细胞中，并且Ala对Aib的全部取代抑制了细胞摄取。含Aib的肽的易位似乎涉及能量不依赖性过程。为了优化作为ODN相互作用部分添加到TV-XIIa的10-mer赖氨酸，将TV-XIIa衍生肽载体的C-末端部分缩短为8-、6-和4-mer Lys。4-和6-mer衍生物不能将ODNs递送到细胞中，另一方面，8-和10-mer衍生物显著促进ODNs的摄取。这些结果表明TV-XIIa的膜透性和Lys残基的数量可能是ODNs进入细胞的重要因素。接下来，以疏水氨基酸Aib和碱性氨基酸Lys为原料，合成了两亲性24-残基螺旋肽[Ac-（U-U-U-K）6-NH 2和Ac-（U-U-K）8-NH 2]。我们发现螺旋肽与ODNs之间通过电相互作用形成的复合物被细胞吸收。

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.