Protamine sulfate enhances the transduction efficiency of recombinant adeno-associated virus-mediated gene delivery.
OBJECTIVE
The purpose of this study was to evaluate glucose responsiveness in HepG2 human hepatoma cells transduced by a recombinant adeno-associated virus (rAAV) vector containing the insulin gene promoter. and to investigate the effect of protamine sulfate on rAAV-mediated gene delivery.


METHODS
Recombinant AAV vector, AAV.Ins.Luc.delta EGFP, was employed to transduce HepG2 hepatoma cells. Virus infection was carried out either in the absence or presence of protamine sulfate, followed by fluorescence microscopic examination, luciferase activity assay, and flow cytometric analysis. Electrokinetic measurements were carried out to determine the effect of protamine sulfate on zeta potential of the cells and the virus.


RESULTS
Glucose-responsive luciferase gene expression was obtained in rAAV-transduced HepG2 cells. Addition of 5 microg/ml protamine reversed the zeta potential of the cells and the virus particles, leading to enhanced transgene expression in rAAV-transduced HepG2 cells. Enhancement of protamine sulfate on rAAV-mediated gene transfer was dose-dependent. Addition of more than 5 microg/ml protamine resulted in a reduction of infectability of the virus.


CONCLUSIONS
Glucose responsiveness in the millimolar concentration range can be obtained in rAAV-transduced HepG2 cells. Protamine sulfate, up to 5 microg/ml, enhanced the rAAV transduction efficiency in HepG2 cells. The enhancement was correlated with zeta potential of the cells and the virus.
Yang
Y W
YW
School of Pharmacy, College of Medicine, National Taiwan University, Taipei. ywyang@ha.mc.ntu.edu.tw
Hsieh
Y C
YC
D004274:DNA, Recombinant / Q000235:genetics / Q000494:pharmacology*; D000229:Dependovirus / Q000187:drug effects* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter / Q000187:drug effects / Q000502:physiology; D005822:Genetic Vectors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006494:Heparin Antagonists / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D011479:Protamines / Q000494:pharmacology*; D014161:Transduction, Genetic / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D004274:DNA, Recombinant; D006494:Heparin Antagonists; D011479:Protamines; D005947:Glucose