Eto Yusuke, Yoshioka Yasuo, Asavatanabodee Ratima, Kida Shinya, Maeda Mitsuko, Mukai Yohei, Mizuguchi Hiroyuki, Kawasaki Koichi, Okada Naoki, Nakagawa Shinsaku
Department of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka 565-0871, Japan.
Peptides. 2009 Aug;30(8):1548-52. doi: 10.1016/j.peptides.2009.05.017. Epub 2009 May 23.
Adenovirus vectors (Advs) are widely used for basic and clinical research because of their high transduction efficiency. However, they are poorly transduced into cells lacking the primary adenovirus receptor, the coxsackievirus and adenovirus receptor (CAR). Here, we generated Adv conjugated with cell-penetrating peptides (CPPs), such as Tat, octaarginine (R8) or proline-rich (Pro) peptides, and compared the transduction properties of these constructs. We constructed the Advs conjugated to the CPPs (CPP-Adv) by chemical conjugation. The CPP-conjugated Advs created with optimal modification ratios led to gene expression 1-2log orders higher than unmodified Adv in CAR-negative cells. Tat-Adv and R8-Adv were taken up into the cells mainly through macropinocytosis, independently of the CAR. In addition, the cellular uptake of Tat-Adv was highly dependent on heparan sulfate on the cell surface, whereas that of R8-Adv was dependent on chondroitin sulfate B. These data suggest that the use of CPP-Advs with different cellular uptake pathways might create new methods for the delivery of Adv. The results obtained in this research encourage the use of CPP-peptide-modified Advs as an attractive tool for transducing cells and as useful platform vectors for gene therapy and basic research.
腺病毒载体（Advs）因其高转导效率而被广泛用于基础和临床研究。然而，它们很难转导到缺乏主要腺病毒受体——柯萨奇病毒和腺病毒受体（CAR）的细胞中。在此，我们生成了与细胞穿透肽（CPPs）如Tat、八聚精氨酸（R8）或富含脯氨酸（Pro）的肽缀合的腺病毒载体，并比较了这些构建体的转导特性。我们通过化学缀合构建了与CPPs缀合的腺病毒载体（CPP-Adv）。以最佳修饰比例构建的与CPP缀合的腺病毒载体在CAR阴性细胞中导致的基因表达比未修饰的腺病毒载体高1-2个对数级。Tat-Adv和R8-Adv主要通过巨胞饮作用进入细胞，与CAR无关。此外，Tat-Adv的细胞摄取高度依赖于细胞表面的硫酸乙酰肝素，而R8-Adv的细胞摄取则依赖于硫酸软骨素B。这些数据表明，使用具有不同细胞摄取途径的CPP-Adv可能会创造腺病毒载体递送的新方法。本研究获得的结果鼓励将CPP肽修饰的腺病毒载体用作转导细胞的有吸引力的工具以及用于基因治疗和基础研究的有用平台载体。
