Incorporation of calcium phosphate enhances recombinant adeno-associated virus-mediated gene therapy in diabetic mice.
BACKGROUND
Increased efficiency of transgene expression is desired for virus-mediated gene delivery. In the present study, we examined the effect of calcium phosphate (CaPi) on recombinant adeno-associated virus (rAAV)-mediated insulin therapy in diabetic animals.


METHODS
The rAAV vector, rAAV.PEPCK.Ins.EGFP, containing the human insulin gene under control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter and the enhanced green fluorescence protein (EGFP) gene driven by the cytomegalovirus (CMV) IE promoter, was employed in this study. C57BL/6J mice were made diabetic with streptozotocin (STZ), followed by injection into the livers with either rAAV alone, or noncovalent complexes with calcium phosphate. Body weight and blood glucose levels of the animals were routinely monitored after 6 h fasting. Secretion of human insulin in the rAAV-transduced animals was determined by radioimmunoassay (RIA). Expression of human insulin in the livers of the animals was detected by immunohistochemical staining.


RESULTS
Compared with the STZ-treated control mice, administration of rAAV containing the human insulin gene significantly decreased blood glucose levels and maintained body weight of the diabetic animals. Complexation of rAAV with calcium phosphate enhanced the hypoglycemic effect of rAAV-mediated gene transfer. Results obtained from both RIA and immunohistochemical staining demonstrated that incorporation of calcium phosphate enhanced rAAV-mediated gene transfer in vivo, leading to higher expression and secretion of human insulin.


CONCLUSIONS
Administration of rAAV harboring the human insulin gene into livers of the STZ-diabetic mice improved blood glucose levels, maintained body weight of the diabetic animals, and resulted in human insulin secretion. Complexation of rAAV with calcium phosphate significantly potentiated the efficiency of rAAV-mediated diabetic gene therapy.
Yang
Ya-Wun
YW
School of Pharmacy, College of Medicine, National Taiwan University, 1 Jen-Ai Road, Section 1, Taipei 100, Taiwan. ywyang@ha.mc.ntu.edu.tw
Chao
Chih-Kai
CK
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D001835:Body Weight; D019556:COS Cells; D002130:Calcium Phosphates / Q000378:metabolism*; D002461:Cell Line, Transformed; D003587:Cytomegalovirus / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics; D011401:Promoter Regions, Genetic; D011863:Radioimmunoassay; D013997:Time Factors
D001786:Blood Glucose; D002130:Calcium Phosphates; D007328:Insulin; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP); C485829:calcium phosphate, dibasic, anhydrous