Role of the oxygen-dependent degradation domain in a hypoxia-inducible gene expression system in vascular endothelial growth factor gene therapy

Honglian Jin, Meng Lu Liu, Hyun Ah Kim, Minhyung Lee, Sungsu An, Jinsoo Oh, Joon Cho, Seong Yi, Keungnyun Kim, Doheum Yoon, Yoon Ha

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Study Design: An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation effect of a reporter or therapeutic gene expression by an oxygen-dependent degradation (ODD) domain in a hypoxia-inducible gene expression system with or without the erythropoietin (EPO) enhancer. Objective: To increase vascular endothelial growth factor (VEGF) gene expression in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer upstream of the SV promoter and an ODD domain C-terminally fused to VEGF. Summary of Background Data: ODD domain plays a major role in the degradation of hypoxia-inducible factor 1α and has been used in a hypoxia-specific gene expression system as a post-translational regulatory factor. Methods: The hypoxia-inducible luciferase or VEGF plasmid was constructed using the EPO enhancer combined with or without the ODD domain. The constructed plasmid was transfected into mouse Neuro 2a (N2a) neuroblastoma cells by Lipofectamine 2000, followed by a 24-hour incubation in hypoxia or normoxia. For in vivo analysis, the naked plasmid DNA was directly injected into the injured rat spinal cord. The gene expression was evaluated by luciferase activity assay, enzyme-linked immunosorbent assay, reverse transcriptase- polymerase chain reaction, and immunofluorescence staining. Results: The EPO enhancer/ODD domain-combined hypoxia-inducible gene expression system clearly increased the expression of the reporter luciferase gene and therapeutic VEGF gene specifically under hypoxic conditions and SCI, and quickly downregulated protein expression to a very low level after reoxygenation. Conclusion: These results strongly suggest the potential applicability of this EPO enhancer/ODD domain-based hypoxia-inducible gene expression system in the development of a safer and more effective VEGF gene therapy for SCI.

Original languageEnglish (US)
JournalSpine
Volume34
Issue number26
DOIs
StatePublished - Dec 1 2009

Fingerprint

Genetic Therapy
Vascular Endothelial Growth Factor A
Oxygen
Gene Expression
Erythropoietin
Spinal Cord Injuries
Luciferases
Plasmids
Hypoxia-Inducible Factor 1
Hypoxia
Reverse Transcriptase Polymerase Chain Reaction
Neuroblastoma
Reporter Genes
Fluorescent Antibody Technique
Spinal Cord
Down-Regulation
Enzyme-Linked Immunosorbent Assay
Staining and Labeling
DNA
Therapeutics

Keywords

  • Gene therapy
  • Hypoxia-inducible gene expression system
  • Oxygen-dependent degradation
  • Spinal cord injury
  • Vascular endothelial growth factor

ASJC Scopus subject areas

  • Orthopedics and Sports Medicine
  • Clinical Neurology

Cite this

Role of the oxygen-dependent degradation domain in a hypoxia-inducible gene expression system in vascular endothelial growth factor gene therapy. / Jin, Honglian; Liu, Meng Lu; Kim, Hyun Ah; Lee, Minhyung; An, Sungsu; Oh, Jinsoo; Cho, Joon; Yi, Seong; Kim, Keungnyun; Yoon, Doheum; Ha, Yoon.

In: Spine, Vol. 34, No. 26, 01.12.2009.

Research output: Contribution to journalArticle

Jin, Honglian ; Liu, Meng Lu ; Kim, Hyun Ah ; Lee, Minhyung ; An, Sungsu ; Oh, Jinsoo ; Cho, Joon ; Yi, Seong ; Kim, Keungnyun ; Yoon, Doheum ; Ha, Yoon. / Role of the oxygen-dependent degradation domain in a hypoxia-inducible gene expression system in vascular endothelial growth factor gene therapy. In: Spine. 2009 ; Vol. 34, No. 26.
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AU - Jin, Honglian

AU - Liu, Meng Lu

AU - Kim, Hyun Ah

AU - Lee, Minhyung

AU - An, Sungsu

AU - Oh, Jinsoo

AU - Cho, Joon

AU - Yi, Seong

AU - Kim, Keungnyun

AU - Yoon, Doheum

AU - Ha, Yoon

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KW - Oxygen-dependent degradation

KW - Spinal cord injury

KW - Vascular endothelial growth factor

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