Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy.
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.
Hsu
Paul Yueh-Jen
PY
School of Pharmacy, College of Medicine, National Taiwan University, 1, Jen-Ai Road, Section 1, Taipei 100, Taiwan.
Yang
Ya-Wun
YW
D000818:Animals; D001835:Body Weight; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000628:therapy*; D004705:Endocytosis; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000235:genetics*; D008099:Liver / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011094:Polyethyleneimine / Q000737:chemistry*
D007328:Insulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine