Premium
Effect of polyethylenimine on recombinant adeno‐associated virus mediated insulin gene therapy
Author(s) -
Hsu Paul YuehJen,
Yang YaWun
Publication year - 2005
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.779
Subject(s) - adeno associated virus , polyethylenimine , genetic enhancement , transgene , insulin , recombinant dna , microbiology and biotechnology , in vivo , reporter gene , biology , transduction (biophysics) , transfection , chemistry , endocrinology , gene expression , gene , vector (molecular biology) , biochemistry
Background 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 . Methods 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. Results 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. Conclusions 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. Copyright © 2005 John Wiley & Sons, Ltd.