Department of General Biology, Medical School, University of Patras, 26504, Rion, Patras, Greece.
Department of Pediatrics, University Hospital of Patras, 26504, Rion, Greece.
Mol Biol Rep. 2019 Jun;46(3):3203-3211. doi: 10.1007/s11033-019-04777-9. Epub 2019 Apr 12.
Liver-directed gene therapy, using mainly viral vectors for the genetic cell modification, is a promising therapeutic approach for many genetic and metabolic liver diseases. The recent successful preclinical trials with AAV vectors expose the benefits as well as the limitations of the system. We focused on the development of an alternative non-viral episomal gene transfer system, by inserting the DNA element Scaffold/Matrix Attachment Region (S/MAR) into the free of antibiotic resistance gene miniplasmid vector (pFAR4). We produced pFAR4 derivative experimental vectors, carrying the eGFP gene driven by the composite HCRHPi liver-specific promoter and either lacking (pFAR4-noS/MAR) or containing the S/MAR element in an upstream (pFAR-S/MAR-IN) or downstream (pFAR4-S/MAR-OUT) configuration in relation to the poly-A signal of the eGFP expression cassette. Upon transfer into Huh7 cells by lipofection, vector pFAR4-S/MAR IN showed significantly higher transfection efficiency and eGFP expression than the control vector or the pFAR4-S/MAR-OUT (p < 0.005), estimated by fluorescent microscopy and flow cytometry. Stable transfections were produced only with cultures containing vector pFAR4-S/MAR IN, through the expansion of single colonies, which displayed sustained GFP expression and plasmid copy number per cell of 2.3 ± 0.4, at 3 months of culture. No vector integration events were detected in these cultures by FISH analysis, while the presence of free, circular plasmids was documented by plasmid rescue assay. The presence of S/MAR renders pFAR4 miniplasmid substantially more efficient regarding episomal gene transfer and is suitable for liver-directed studies towards gene therapy applications.
肝靶向基因治疗主要采用病毒载体进行遗传细胞修饰，是许多遗传和代谢性肝病的有前途的治疗方法。最近使用 AAV 载体进行的成功临床前试验揭示了该系统的优势和局限性。我们专注于开发替代的非病毒附加体基因转移系统，方法是将 DNA 元件支架/基质附着区 (S/MAR) 插入不含抗生素抗性基因的小型质粒载体 (pFAR4) 中。我们生产了携带 eGFP 基因的 pFAR4 衍生实验载体，该基因由复合 HCRHPi 肝特异性启动子驱动，要么缺乏 S/MAR 元件（pFAR4-noS/MAR），要么在 eGFP 表达盒的 poly-A 信号上游（pFAR-S/MAR-IN）或下游（pFAR4-S/MAR-OUT）位置包含 S/MAR 元件。通过脂质体转染将载体转入 Huh7 细胞后，载体 pFAR4-S/MAR IN 显示出比对照载体或 pFAR4-S/MAR-OUT 显著更高的转染效率和 eGFP 表达（p < 0.005），通过荧光显微镜和流式细胞术估计。只有在含有载体 pFAR4-S/MAR IN 的培养物中才能产生稳定的转染，通过单细胞集落的扩增，这些细胞显示出持续的 GFP 表达和每个细胞的质粒拷贝数为 2.3 ± 0.4，在 3 个月的培养中。通过 FISH 分析未在这些培养物中检测到载体整合事件，而通过质粒拯救测定证明存在游离的环状质粒。S/MAR 的存在使 pFAR4 小型质粒在附加体基因转移方面效率显著提高，适合用于肝靶向基因治疗应用的研究。
