Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
Biochem Biophys Res Commun. 2010 Jan 29;392(1):9-15. doi: 10.1016/j.bbrc.2009.12.135. Epub 2009 Dec 30.
Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 microg of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.
非病毒基因传递是一种安全且合适的替代病毒载体介导的传递方法，可以克服与病毒载体相关的免疫原性和致瘤性。我们利用新型的、源自人类的 Hph-1 蛋白转导结构域，该结构域可以促进蛋白进入细胞，开发了一种新的策略，用于体外和体内传递裸露的 DNA。新的 DNA 传递系统包含 Hph-1-GAL4 DNA 结合域（DBD）融合蛋白和增强型绿色荧光蛋白（EGFP）报告质粒，该质粒包含五个重复的 GAL4 上游激活序列（UAS）。Hph-1-GAL4-DBD 蛋白通过 GAL4-DBD 和 UAS 之间的特异性相互作用与质粒 DNA 形成复合物，并通过 Hph-1-PTD 递送至细胞内。pEGFP DNA 成功地被 Hph-1-GAL4 系统传递，并且 EGFP 在哺乳动物细胞（如 HeLa 和 Jurkat）以及 Bright Yellow-2（BY-2）植物细胞中得到有效表达。当使用 Hph-1-GAL4 蛋白将 10μg 的 pEGFP DNA 经鼻腔给予小鼠时，在肺部组织中检测到 EGFP 的高水平表达持续了 7 天。这些结果表明，Hph-1-PTD 介导的 DNA 传递策略可能是一种用于基因治疗和 DNA 疫苗的有用的非病毒 DNA 传递系统。
