Yoshioka Yasuo, Asavatanabodee Ratima, Eto Yusuke, Watanabe Hikaru, Morishige Tomohiro, Yao Xinglei, Kida Shinya, Maeda Mitsuko, Mukai Yohei, Mizuguchi Hiroyuki, Kawasaki Koichi, Okada Naoki, Nakagawa Shinsaku
The center for Advanced Medical Engineering and Informatics, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
Life Sci. 2008 Nov 21;83(21-22):747-55. doi: 10.1016/j.lfs.2008.09.022. Epub 2008 Oct 5.
Adenovirus vectors (Advs) have been very useful for basic research and clinical gene therapy because they propagate to high titers and efficiently transduce cells and tissues regardless of the mitotic status. However, poor transduction of cells that lack the coxsackievirus and adenovirus receptor (CAR), the primary receptor for Advs, has limited Adv application. In this study, we attempted to generate novel Tat-Advs (Advs conjugated with the HIV Tat-derived peptide, a protein-transduction domain (PTD)) to broaden Adv tropism and enhance transduction efficiency.
We constructed Tat-Advs by chemically conjugating Tat peptide to the surface-exposed lysine residues on Advs. We compared the gene transfer activity of Tat-Advs with that of unmodified Advs by measuring the luciferase expression in several types of cell lines.
Tat-Advs showed gene expression 1 to 3 log orders higher than unmodified Advs in CAR-negative adherent cells and blood cells, which are refractory to conventional Advs. The inhibition of Tat-Adv-mediated gene expression by heparin and macropinocytosis inhibitor confirms that binding of Tat-Adv to cellular HSPGs and macropinocytosis are essential for efficient CAR-independent transduction. We also demonstrated that Adv modified with another PTD (R8) had the same high transduction efficiency as Tat-Adv.
These data suggest that Tat-Advs are important tools for transducing cells and will be useful as platform vectors for gene therapy.
腺病毒载体（Advs）对于基础研究和临床基因治疗非常有用，因为它们能高效繁殖，且无论细胞的有丝分裂状态如何，都能有效地转导细胞和组织。然而，缺乏柯萨奇病毒和腺病毒受体（CAR）（Advs的主要受体）的细胞转导效率低下，限制了Advs的应用。在本研究中，我们试图构建新型的Tat-Advs（与HIV Tat衍生肽（一种蛋白质转导结构域（PTD））偶联的Advs），以拓宽Advs的嗜性并提高转导效率。
我们通过将Tat肽化学偶联到Advs表面暴露的赖氨酸残基上来构建Tat-Advs。通过测量几种细胞系中的荧光素酶表达，我们比较了Tat-Advs与未修饰的Advs的基因转移活性。
在CAR阴性贴壁细胞和血细胞中，Tat-Advs的基因表达比未修饰的Advs高1至3个对数级，而这些细胞对传统Advs具有抗性。肝素和巨胞饮作用抑制剂对Tat-Advs介导的基因表达的抑制作用证实，Tat-Advs与细胞硫酸乙酰肝素蛋白聚糖的结合以及巨胞饮作用对于高效的不依赖CAR的转导至关重要。我们还证明，用另一种PTD（R8）修饰的Advs具有与Tat-Advs相同的高转导效率。
这些数据表明，Tat-Advs是转导细胞的重要工具，将作为基因治疗的平台载体发挥作用。
