School of Pharmacy, Zhengzhou University, Zhengzhou, China.
Eur J Pharm Biopharm. 2011 Aug;78(3):320-5. doi: 10.1016/j.ejpb.2010.12.036. Epub 2011 Jan 8.
The purpose of this study is to explore the possibility and feasibility of liver-specific gene therapy. A shRNA expression plasmid against human telomerase reverse transcriptase (hTERT) was constructed under the control of liver-specific promoter apolipoprotein A-I (ApoAI), designated as pApoAI-shTERT, and its liver-specific cytotoxicity and inhibition of telomerase activity were first evaluated in different cell lines, and its therapeutic effect was further studied in SMMC-7721 human liver tumor-bearing mice in vivo. The results showed that compared to pU6-shTERT, a shRNA expression plasmid against hTERT under the control of U6 promoter, pApoAI-shTERT only significantly diminished the cell viability in the telomerase positive hepatocarcinoma cells and showed no cytotoxicity in the telomerase negative cell lines as well as in the telomerase positive cell line of non-liver origin. Besides, pApoAI-shTERT only significantly reduced telomerase activity in the telomerase positive cell lines of liver origin. Intravenous administration of pegylated immuno-lipopolyplexes (PILP) formulated green fluorescent protein (GFP) expression plasmid under the control of ApoAI into liver tumor-bearing mice resulted in restricted GFP expression in liver and liver tumor. The treatment of pApoAI-shTERT formulated as PILP caused a 56% increase in the life span of SMMC-7721 tumor-bearing mice in vivo relative to the control, which was in agreement with the reduced tumor size and down-regulated hTERT mRNA level in the tumors. We conclude that the vector pApoAI-shTERT was able to cause liver-specific and hTERT target-specific cytotoxicity, and utilizing PILP to deliver pApoAI-shTERT is a promising strategy for liver-specific gene therapy.
本研究旨在探讨肝特异性基因治疗的可能性和可行性。构建了一种在载脂蛋白 A-I（ApoAI）启动子控制下表达靶向人端粒酶逆转录酶（hTERT）的短发夹 RNA（shRNA）表达质粒，命名为 pApoAI-shTERT，并首先在不同细胞系中评价其肝特异性细胞毒性和对端粒酶活性的抑制作用，然后在体内 SMMC-7721 人肝癌荷瘤小鼠中进一步研究其治疗效果。结果表明，与 U6 启动子控制下的靶向 hTERT 的 shRNA 表达质粒 pU6-shTERT 相比，pApoAI-shTERT 仅显著降低端粒酶阳性肝癌细胞的细胞活力，而在端粒酶阴性细胞系以及非肝来源的端粒酶阳性细胞系中无细胞毒性。此外，pApoAI-shTERT 仅显著降低肝来源的端粒酶阳性细胞系中的端粒酶活性。将载脂蛋白 A-I 控制下的绿色荧光蛋白（GFP）表达质粒的聚乙二醇化免疫脂质体（PILP）静脉注射到肝癌荷瘤小鼠体内，导致 GFP 在肝和肝肿瘤中的表达受限。PILP 包载的 pApoAI-shTERT 治疗使 SMMC-7721 肝癌荷瘤小鼠的生存期相对于对照组延长了 56%，这与肿瘤体积缩小和 hTERT mRNA 水平下调一致。我们得出结论，载体 pApoAI-shTERT 能够引起肝特异性和 hTERT 靶特异性细胞毒性，利用 PILP 递送 pApoAI-shTERT 是肝特异性基因治疗的一种有前途的策略。
