Development of peptide vectors containing Aid residues for gene therapy

用于基因治疗的含有Aid残基的肽载体的开发

• 批准号: 18590111
• 负责人: WADA Shun-ichi
• 金额: $ 2.45万
• 依托单位: Osaka University of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590111/
• 关键词: Peptide-derived vector; DNA; oligonucleotide; α-aminoisobutyric acid; Aib; peptaibol

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.

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)]，在 TV-XIIa 和 10 聚体赖氨酸 (Lys) 之间缀合，可以传递20聚体寡核苷酸（ODN）进入细胞，并且单独的Lys的10聚体不能递送它们然后，TV-XIIa本身被认为在递送系统中发挥重要作用以获得细胞摄取的直接证据。为了研究TV-XIIa本身并研究20聚体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的肽并将其应用于活细胞以直接观察TV-XIIa的行为此外，为了了解序列中三个不寻常的 Aib 残基的重要性，将它们替换为 Ala，并且还检查了 Aib→Ala 替代类似物在活细胞中的行为。荧光素标记的 TV-XIIa 肽可以易位到细胞中，并且 Aib 全部替换为 Ala 会抑制细胞摄取。含 Aib 的肽的易位似乎涉及能量非依赖性。为了优化作为 ODN 相互作用部分添加到 TV-XIIa 的 10 聚体赖氨酸，TV-XIIa 衍生肽载体的 C 端部分被缩短为赖氨酸的 8 聚体、6 聚体和 4 聚体。 4 聚体和 6 聚体衍生物不能将 ODN 递送到细胞中，而 8 聚体和 10 聚体衍生物则显着促进 ODN 的摄取。表明 TV-XIIa 的膜渗透性和 Lys 残基的数量可能是将 ODN 递送到细胞中的重要因素接下来，两亲性 24 残基螺旋肽 [Ac-(U-U-U-K)6-NH2 和 Ac-(U-U-K)8- NH2]是使用疏水性氨基酸Aib和碱性氨基酸Lys合成的，我们发现这些复合物是通过形成的。螺旋肽和 ODN 之间的电相互作用被吸收到细胞中。

项目成果

Wada Membrane permeability of Aib-containing peptide, TV-XIIa and its derivative

含 Aib 肽、TV-XIIa 及其衍生物的和田膜渗透性

• 发表时间: 2008
• 作者: Shun-ichi Wada;Yasunari Hitora;Reiko Tanaka;Hidehito Urata;和田 俊一;Shun-ichi Wada and Reiko Tanaka;Shun-ichi Wada(和田俊一);和田 俊一;Shun-ichi
• 通讯作者: Shun-ichi

Aib含有ペプチドを用いたオリゴヌクレオチドの細胞内導入

使用含有 Aib 的肽将寡核苷酸引入细胞内

• 发表时间: 2007
• 作者: Shun-ichi Wada;Yasunari Hitora;Reiko Tanaka;Hidehito Urata;和田 俊一;Shun-ichi Wada and Reiko Tanaka;Shun-ichi Wada(和田俊一);和田 俊一;Shun-ichi;和田 俊一;和田 俊一;Shun-ichi;和田 俊一;和田 俊一
• 通讯作者: 和田 俊一

Functionalized 20-Residual Peptaibol for Nucleic Acid Delivery

用于核酸输送的功能化 20 残基 Peptaibol

• 发表时间: 2007
• 期刊: Chemistry & Biodiversity 4
• 作者: Shun-ichi Wada;Yasunari Hitora;Reiko Tanaka;Hidehito Urata;和田 俊一
• 通讯作者: 和田 俊一

Translocation of an Aib-containing Peptide through Cell Membranes

含 Aib 的肽通过细胞膜的易位

• 发表时间: 2008
• 期刊: Bioorganic & Medicinal Chemistry Letters (印刷中)
• 作者: 吉村 祐一;浅見 和弘;高畑 廣紀;吉村祐一,山崎佳子,浅見和弘,高畑廣紀;吉村祐一,和知克典,山崎佳子,高畑廣紀;山崎佳子,吉村祐一,高畑廣紀;吉村祐一,松井祐充,田中博道,高畑廣紀;和田 俊一
• 通讯作者: 和田 俊一

Aib含有ペプチドの膜透過性に関する研究

含Aib肽的膜通透性研究

• 发表时间: 2007
• 作者: Shun-ichi Wada;Yasunari Hitora;Reiko Tanaka;Hidehito Urata;和田 俊一;Shun-ichi Wada and Reiko Tanaka;Shun-ichi Wada(和田俊一);和田 俊一;Shun-ichi;和田 俊一;和田 俊一
• 通讯作者: 和田 俊一