Hsu Paul Yueh-Jen, Yang Ya-Wun
School of Pharmacy, College of Medicine, National Taiwan University, 1, Jen-Ai Road, Section 1, Taipei 100, Taiwan.
J Gene Med. 2005 Oct;7(10):1311-21. doi: 10.1002/jgm.779.
Recombinant adeno-associated virus (rAAV) is becoming a promising vector for gene therapy for type I diabetes. The objective of this study was to investigate the effect of incorporation of polyethylenimine (PEI) on rAAV-mediated insulin gene therapy in vitro and in vivo.
Recombinant AAV vector, harboring the furin-mutated human insulin and enhanced green fluorescent protein (EGFP) genes, was constructed. The effect of complexation with PEI on rAAV-mediated gene transfer was examined in Huh7 human hepatoma cells. The transgene expression was also examined in streptozotocin (STZ)-induced diabetic C57BL/6J mice by direct administration of rAAV into the livers of the animals, followed by monitoring changes in body weight and blood glucose levels. Secretion of human insulin was determined by radioimmunoassay (RIA) and immunohistochemical staining in the livers.
Complexation with PEI was shown to enhance rAAV-mediated transgene expression in Huh7 cells, resulting in higher transduction efficiency and enhanced production of immunoreactive human insulin. Heparin competition assay demonstrated that endocytosis of rAAV-PEI was partially inhibited by heparin. The enhancement of rAAV-mediated transgene expression was also demonstrated in the animals, showing lowering of blood glucose and longer duration of normoglycemia. Immunofluorescent staining of the liver sections demonstrated that PEI increased the uptake of rAAV and enhanced insulin secretion. The enhancement of PEI on rAAV-mediated insulin gene therapy was further confirmed by glucose challenge and a 10-h fasting blood glucose test.
Results obtained in this study demonstrated that incorporation of PEI augmented rAAV-mediated insulin gene transfer and enhanced amelioration of hyperglycemia in the STZ-induced diabetic animals.
重组腺相关病毒（rAAV）正成为I型糖尿病基因治疗的一种有前景的载体。本研究的目的是探讨聚乙烯亚胺（PEI）的掺入对rAAV介导的体外和体内胰岛素基因治疗的影响。
构建携带弗林蛋白酶突变的人胰岛素和增强型绿色荧光蛋白（EGFP）基因的重组AAV载体。在Huh7人肝癌细胞中检测与PEI复合对rAAV介导的基因转移的影响。通过将rAAV直接注射到链脲佐菌素（STZ）诱导的糖尿病C57BL/6J小鼠肝脏中，然后监测动物体重和血糖水平的变化，来检测转基因表达。通过放射免疫测定（RIA）和肝脏免疫组织化学染色测定人胰岛素的分泌。
与PEI复合显示可增强rAAV介导的Huh7细胞中转基因表达，导致更高的转导效率和增强的免疫反应性人胰岛素产生。肝素竞争试验表明，肝素部分抑制了rAAV-PEI的内吞作用。在动物中也证明了rAAV介导的转基因表达增强，表现为血糖降低和正常血糖持续时间延长。肝脏切片的免疫荧光染色表明，PEI增加了rAAV的摄取并增强了胰岛素分泌。葡萄糖激发试验和10小时空腹血糖试验进一步证实了PEI对rAAV介导的胰岛素基因治疗的增强作用。
本研究获得的结果表明，PEI的掺入增强了rAAV介导的胰岛素基因转移，并增强了STZ诱导的糖尿病动物高血糖的改善。
