Stable expression of the oxc and frc genes from Oxalobacter formigenes in human embryo kidney 293 cells: implications for gene therapy of hyperoxaluria.
Hyperoxaluria can lead to multiple pathologic conditions such as recurrent urolithiasis, oxalosis, nephrocalcinosis and even renal failure, but there is no known oxalate-degrading pathway in the human body, and current therapeutic options for patients with hyperoxaluria are limited. Oxalyl-CoA decarboxylase and formyl-CoA transferase are the key enzymes in the oxalate catabolism of Oxalobacter formigenes which dwell in the intestine of vertebrates and have an important symbiotic relationship with their hosts. The aim of this study was to insert the oxalate-degrading enzyme genes into human embryo kidney (HEK) 293 cells and to evaluate if the oxalate-degrading enzymes could be expressed in these cells and keep their enzyme activity. We present here the cloning of the two genes from O. formigenes and their subsequent expression in HEK293 cells. The results showed that the expression of oxalyl-CoA decarboxylase and formyl-CoA transferase was confirmed by RT-PCR and Western blotting, and the proteins were located in the cytoplasm of transfected cells. Furthermore, the transfected cells were capable to a certain degree of degrading oxalate. These findings suggest that the transfer of oxalate-degrading enzyme genes into human cells is possibly a potential candidate for the gene therapy of hyperoxaluria.
Ye
Zhang-Qun
ZQ
Department of Urology, Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
Kong
De-Bo
DB
Chen
Zhi-Qiang
ZQ
Yao
Lin-Fang
LF
Guo
Hui
H
Yu
Xiao
X
Liu
Guan-Lin
GL
Yang
Wei-Min
WM
D000214:Acyl Coenzyme A / Q000378:metabolism; D002460:Cell Line; D003065:Coenzyme A / Q000378:metabolism; D003653:Decarboxylation; D005453:Fluorescence; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006959:Hyperoxaluria / Q000628:therapy*; D010070:Oxalates / Q000378:metabolism; D020624:Oxalobacter formigenes / Q000235:genetics*; D010957:Plasmids; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection