Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system.
METHODS
A spinal cord injury and in vitro neural hypoxia models were used to evaluate the hypoxia responsive gene expression.


OBJECTIVE
To limit the risk of unwanted overexpression of therapeutic genes, we developed a hypoxia-inducible gene therapy system using the erythropoietin (Epo) enhancer and the RTP801 promoter.


BACKGROUND
Gene therapy is an emerging therapeutic technique to treat spinal cord injury. However, uncontrolled overexpression of therapeutic genes in nondisease tissues during gene therapy raises a doubt about its safety. Post-traumatic ischemia is an important factor worsening the spinal cord damage, and hypoxia could regulate the gene expressions using a hypoxia-inducible promoter.


METHODS
The plasmids, pEpo-SV-Luc and pRTP801-Luc, were constructed. Mouse neuroblastoma cells (N2A) were used to evaluate the hypoxia-inducible gene expression in vitro.- Gene transfection and expression were allowed for 24 hours under normoxia (pO2, 152 mm Hg) or hypoxia (pO2, 7.6 mm Hg). Spinal cord injury was made using clip compression. Plasmids were injected directly into the injured spinal cord immediately following injury. The gene expression was assessed by luciferase assay.


RESULTS
pEpo-SV-Luc and pRTP801-Luc showed more than three times higher gene expression in N2A cells under hypoxia than normoxia. The expression level of luciferase in the injured spinal cord was higher than in the normal spinal cord. Immunostaining demonstrated that neurons, astrocytes, and capillary endothelial cells expressed luciferase in the cytoplasm.


CONCLUSIONS
The pEpo-SV-Luc and pRTP801-Luc systems are effective in that they induce gene expression specifically in neurons under the hypoxic condition and spinal cord injury.
Lee
Minhyung
M
Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea.
Lee
Eun Su
ES
Kim
Young Soo
YS
Choi
Byung Hyune
BH
Park
So Ra
SR
Park
Hyun Seon
HS
Park
Hyung Chun
HC
Kim
Sung Wan
SW
Ha
Yoon
Y
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000235:genetics; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000502:physiology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D051379:Mice; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries / Q000235:genetics / Q000378:metabolism*; D020760:Spinal Cord Ischemia / Q000235:genetics / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D008156:Luciferases