Nguyen Andrew T, Dow Adrienne C, Kupiec-Weglinski Jerzy, Busuttil Ronald W, Lipshutz Gerald S
Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095-7054, USA.
J Surg Res. 2008 Jul;148(1):60-6. doi: 10.1016/j.jss.2008.02.016. Epub 2008 Mar 13.
Gene therapy represents a promising treatment for hepatic disease. Most approaches today use viral methods to target tissues. While nonviral gene therapy is less prominent, hydrodynamic gene delivery represents a promising approach to direct gene expression to the liver. The purpose of the present study was to evaluate promoters for efficient gene expression in hepatocytes in vivo by hydrodynamic delivery and to test the findings in a model of hemophilia A.
Human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), elongation factor-1 alpha (EF1alpha), and phosphoglycerokinase (PGK) promoters were subcloned into plasmids with a luciferase reporter gene. In vitro calcium phosphate-mediated transfection of 2 x 10(5) HEK 293 cells was followed by in vivo whole animal bioluminescence and luminometry after hydrodynamic tail vein injection of plasmid DNA. Six-month-old FVB factor VIII (FVIII)-deficient mice were similarly injected with CBA- or EF1alpha-promoted constructs containing the FVIII heavy and light chains and expression was examined.
In vitro transfection demonstrated a hierarchy of expression: hCMV-intron>CAG>EF1alpha>hCMV>>PGK. In vivo luminometry demonstrated that the CAG construct produced 2.6x, 3.0x, 3.4x, and >1000x the expression of the hCMV-intron, EF1alpha, hCMV, and PGK constructs respectively. FVIII plasmid injected hemophilic mice demonstrated higher levels of FVIII expression with CAG versus EF1alpha, confirming the reporter gene studies. All FVIII-deficient mice injected with EF1alpha-FVIII or CAG-FVIII plasmids survived after tail clipping.
The CAG promoter/enhancer combination is an excellent alternative to the human CMV promoter for hydrodynamic gene delivery to the liver.
基因治疗是肝病的一种有前景的治疗方法。目前大多数方法使用病毒方法来靶向组织。虽然非病毒基因治疗不太突出，但流体动力学基因递送是一种将基因表达直接导向肝脏的有前景的方法。本研究的目的是通过流体动力学递送评估用于体内肝细胞高效基因表达的启动子，并在甲型血友病模型中验证这些发现。
将人巨细胞病毒（hCMV）、鸡β-肌动蛋白/CMV增强子（CAG）、延伸因子-1α（EF1α）和磷酸甘油酸激酶（PGK）启动子亚克隆到带有荧光素酶报告基因的质粒中。对2×10⁵ HEK 293细胞进行体外磷酸钙介导的转染，然后在通过尾静脉流体动力学注射质粒DNA后进行体内全动物生物发光和发光测定。对6月龄的FVB因子VIII（FVIII）缺陷小鼠同样注射含有FVIII重链和轻链的CBA或EF1α启动子构建体，并检测表达情况。
体外转染显示出表达层次：hCMV-内含子>CAG>EF1α>hCMV>>PGK。体内发光测定表明，CAG构建体产生的表达分别是hCMV-内含子、EF1α、hCMV和PGK构建体的2.6倍、3.0倍、3.4倍和>1000倍。注射FVIII质粒的血友病小鼠显示，与EF1α相比，CAG的FVIII表达水平更高，证实了报告基因研究结果。所有注射EF1α-FVIII或CAG-FVIII质粒的FVIII缺陷小鼠在剪尾后均存活。
CAG启动子/增强子组合是用于流体动力学基因递送至肝脏的人CMV启动子的极佳替代品。
