Controlled nonviral gene delivery and expression using stable neural stem cell line transfected with a hypoxia-inducible gene expression system
✍ Scribed by Meng-Lu Liu; Jin Soo Oh; Sung Su An; William A. Pennant; Hyo Jin Kim; So-Jung Gwak; Do Heum Yoon; Keung Nyun Kim; Minhyung Lee; Yoon Ha
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 483 KB
- Volume
- 12
- Category
- Article
- ISSN
- 1099-498X
- DOI
- 10.1002/jgm.1527
No coin nor oath required. For personal study only.
✦ Synopsis
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 hypoxiaregulated 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 hypoxiaregulated luciferase expression after differentiation.
Conclusions
We propose that NSCs engineered with the Epo-SVtherapeutic 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.