Abbaspour Akbar, Esmaeilzadeh Abdolreza, Sharafi Ali
Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
Department of Pharmaceutical Biomaterials, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran.
3 Biotech. 2021 Mar;11(3):140. doi: 10.1007/s13205-021-02692-0. Epub 2021 Feb 24.
In this study, a suicide gene therapy approach was optimized by a non-viral polyplex system based on pEGFP-N1 vector harboring purine nucleoside phosphorylase gene conducted by vascular endothelial growth factor promoter for an in vitro breast cancer model (4T1 cell line). The VEGF promoter and purine nucleoside phosphorylase gene were cloned into the vector from the source of 4T1 and genomic DNA, respectively. A gene construct was developed by replacing VEGF promoter instead of CMV promoter in pEGFP-N1vector. PNP gene was integrated in to the multiple cloning site of the obtained vector. On the other hand, a construct from pEGFP-N1 harboring PNP gene under the control of the original CMV promoter was developed. The transfection method using cationic polymer was optimized based on N/P ratio, cell cytotoxicity, polyplex size, potential and the green fluorescent protein (GFP) expression by fluorescent microscopy and flowcytometry. Also, the effect of hypoxia condition induced by 0.5 mM HO on the promoter efficiency was investigated. The results showed that the performed gene delivery system is capable of the gene transfection to more than 30% of the cancer cells with both VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1 plasmids. The hypoxia condition did not show a significant effect on the VEGF promoter. But, it revealed that bystander effect can improve the efficacy of this system and reduce drug IC50 to 2 and fourfold for plasmids VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1, respectively. These results showed that the bystander effect could almost compensate the low efficiency of non-viral gene delivery systems. We suggest that the tumor-specific gene expression system mediated by the VEGF promoter can be especially useful in the present model of breast cancer gene therapy.
在本研究中，基于携带嘌呤核苷磷酸化酶基因的pEGFP-N1载体的非病毒多聚体系统，通过血管内皮生长因子启动子对体外乳腺癌模型（4T1细胞系）优化了自杀基因治疗方法。VEGF启动子和嘌呤核苷磷酸化酶基因分别从4T1和基因组DNA来源克隆到载体中。通过在pEGFP-N1载体中替换VEGF启动子而非CMV启动子构建了一种基因构建体。将PNP基因整合到所得载体的多克隆位点。另一方面，构建了在原始CMV启动子控制下携带PNP基因的pEGFP-N1构建体。基于N/P比、细胞毒性、多聚体大小、电位以及通过荧光显微镜和流式细胞术检测的绿色荧光蛋白（GFP）表达，优化了使用阳离子聚合物的转染方法。此外，研究了0.5 mM HO诱导的缺氧条件对启动子效率的影响。结果表明，所实施的基因递送系统能够用VEGF-PNP-pEGFP-N1和PNP-pEGFP-N1质粒将基因转染到超过30%的癌细胞中。缺氧条件对VEGF启动子未显示出显著影响。但是，结果表明旁观者效应可提高该系统的疗效，并使VEGF-PNP-pEGFP-N1和PNP-pEGFP-N1质粒的药物IC50分别降低2倍和4倍。这些结果表明旁观者效应几乎可以弥补非病毒基因递送系统的低效率。我们认为，由VEGF启动子介导的肿瘤特异性基因表达系统在目前的乳腺癌基因治疗模型中可能特别有用。
