title: Controlled nonviral gene delivery and expression using stable neural stem cell line transfected with a hypoxia-inducible gene expression system.
abstract: BACKGROUND
  Nonviral ex vivo local gene therapy systems consisting of regulated gene expression vectors and cellular delivery platforms represent a novel strategy for tissue repair and regeneration. We introduced a hypoxia-regulated plasmid-based system into mouse neural stem cells (NSCs) as an efficient gene expression and delivery platform for rapid, robust and persistent hypoxic/ischemic-regulated gene expression in the spinal cord.
  
  
  METHODS
  A synthetic hypoxia-responsive erythropoietin (Epo) enhancer, the SV40 minimal promoter and the luciferase (Luc) reporter gene were incorporated in a DsRed-expressing double-promoter plasmid for cell lipofection and Zeocin-selection to establish a hypoxia-regulated stable NSC line (NSC-Epo-SV-Luc). A nonhypoxia-regulated stable NSC line (NSC-SV-Luc) was also established as a control.
  
  
  RESULTS
  Under the transcriptional regulation of the Epo enhancer, in vitro luciferase expression in NSC-Epo-SV-Luc, but not in NSC-SV-Luc, was sensitively augmented according to the strength and duration of the hypoxic stimulus and was quickly down-regulated to a low basal level after reoxygenation of the hypoxic cells. Furthermore, deoxygenation of the reoxygenated cells clearly enhanced the luciferase activity again. After transplantation into a rat spinal cord injury (SCI) model, only NSC-Epo-SV-Luc showed ischemic injury-specific luciferase expression Notably, the engineered NSC lines kept the neural differentiation potential and retained the hypoxia-regulated luciferase expression after differentiation.
  
  
  CONCLUSIONS
  We propose that NSCs engineered with the Epo-SV-therapeutic gene will be valuable for developing a controllable stem cell-mediated nonviral gene therapy for SCI or other central nervous system diseases accompanied with chronic or episodic hypoxic/ischemic stresses.
authors.affiliation: Department of Neurosurgery, Spine & Spinal Cord Institute, College of Medicine, Yonsei University, Seoul, Korea.
authors.forename: Meng-Lu
authors.initials: ML
authors.lastname: Liu
authors.forename: Jin Soo
authors.initials: JS
authors.lastname: Oh
authors.forename: Sung Su
authors.initials: SS
authors.lastname: An
authors.forename: William A
authors.initials: WA
authors.lastname: Pennant
authors.forename: Hyo Jin
authors.initials: HJ
authors.lastname: Kim
authors.forename: So-Jung
authors.initials: SJ
authors.lastname: Gwak
authors.forename: Do Heum
authors.initials: DH
authors.lastname: Yoon
authors.forename: Keung Nyun
authors.initials: KN
authors.lastname: Kim
authors.forename: Minhyung
authors.initials: M
authors.lastname: Lee
authors.forename: Yoon
authors.initials: Y
authors.lastname: Ha
mesh_terms: D000818:Animals; D002460:Cell Line; D004921:Erythropoietin; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D000860:Hypoxia; D051379:Mice; D058953:Neural Stem Cells / Q000378:metabolism / Q000637:transplantation*; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D004921:Erythropoietin