A hypoxia-inducible gene expression system using erythropoietin 3' untranslated region for the gene therapy of rat spinal cord injury.
Many neurologic disorders are accompanied by ischemic injury during the pathologic process. To develop a controllable and injury-specific gene therapy system for the neurologic disorders, we constructed a hypoxia inducible plasmid with the erythropoietin (Epo) 3' untranslated region (UTR), which can enhance the stability of target mRNAs in response to hypoxia. The Epo 3' UTR was inserted at the 3' flanking region of luciferase gene in pSV-Luc, resulting in the construction of pSV-Luc-EpoUTR. In pEpo-SV-Luc-EpoUTR, the Epo enhancer was inserted into the upstream of the SV40 promoter to increase the hypoxia inducibility. The plasmids were evaluated in N2a mouse neuroblastoma cells under hypoxic conditions and in a rat spinal cord injury (SCI) model. The results showed that the Epo 3' UTR alone showed a three-fold increase in luciferase activity in hypoxic N2a cells as well as in the rat SCI model when compared to the sham control. In contrast, the Epo 3' UTR showed no effect on the luciferase activity in the presence of the Epo enhancer, probably because the Epo enhancer was more sensitive to hypoxia and showed a dominant effect. However, the Epo enhancer itself showed high level of luciferase activity even in normoxia (about five to eight-folds increase), while the Epo 3' UTR did not show enhanced background activity. Immunohistochemical staining showed expression of luciferase from pSV-Luc-EpoUTR both in neurons and astrocytes around the injured spinal cord of rat. These results suggest that the Epo 3' UTR could provide a specific and safe system for the hypoxia-inducible gene therapy of the neurologic disorders including SCI.
Choi
Byung Hyune
BH
Inha Research Institute for Medical Sciences, Inha University College of Medicine, Incheon, South Korea.
Ha
Yoon
Y
Ahn
Cheol-Hee
CH
Huang
Xian
X
Kim
Jin-Mo
JM
Park
So Ra
SR
Park
Hyeonseon
H
Park
Hyung Chun
HC
Kim
Sung Wan
SW
Lee
Minhyung
M
D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D015687:Cell Hypoxia / Q000235:genetics*; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D004742:Enhancer Elements, Genetic / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000627:therapeutic use; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013539:Simian virus 40 / Q000235:genetics; D013119:Spinal Cord Injuries / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D020760:Spinal Cord Ischemia / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D015854:Up-Regulation / Q000235:genetics
D020413:3' Untranslated Regions; D004921:Erythropoietin; D008156:Luciferases