Skip to main content

Advertisement

SpringerLink
Log in

An Insult-Inducible Vector System Activated by Hypoxia and Oxidative Stress for Neuronal Gene Therapy

  • Published:
Translational Stroke Research Aims and scope Submit manuscript

Abstract

Gene therapy has demonstrated the protective potential of a variety of genes against stroke. However, conventional gene therapy vectors are limited due to the inability to temporally control their expression, which can sometimes lead to deleterious side effects. Thus, an inducible vector that can be temporally controlled and activated by the insult itself would be advantageous. Using hypoxia responsive elements (HRE) and antioxidant responsive elements (ARE), we have constructed an insult-inducible vector activated by hypoxia and reactive oxygen species (ROS). In COS7 cells, the inducible ARE–HRE-luciferase vectors are highly activated by oxygen deprivation, hydrogen peroxide treatment, and the ROS-induced transcription factor NF-E2-related factor 2 (Nrf2). Using a defective herpes virus, the neuroprotective potential of this inducible vector was tested by over-expressing the transcription factor Nrf2. In primary cortical cultures, expression of the inducible ARE–HRE–Nrf2 protects against oxygen glucose deprivation, similar to that afforded by the constitutively expressed Nrf2. This ARE + HRE vector system is advantageous in that it allows the expression of a transgene to be activated not only during hypoxia but also maintained after reperfusion, thus prolonging the transgene expression during an ischemic insult. This insult-inducible vector system will be a valuable gene therapy tool for activating therapeutic/protective genes in cerebrovascular diseases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol. 2000;884:1474–80.

    Google Scholar 

  2. Sharp FR, Bernaudin M. HIF1 and oxygen sensing in the brain. Nat Rev. 2004;56:437–48.

    Google Scholar 

  3. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA. 1995;9212:5510–14.

    Article  Google Scholar 

  4. Semenza GL. Hypoxia-inducible factor 1: control of oxygen homeostasis in health and disease. Pediatr Res. 2001;495:614–17.

    Article  Google Scholar 

  5. Lee JM, Zipfel GJ, Choi DW. The changing landscape of ischaemic brain injury mechanisms. Nature. 1999;3996738(Suppl):A7–14.

    Google Scholar 

  6. Sapolsky RM. Neuroprotective gene therapy against acute neurological insults. Nat Rev. 2003;41:61–9.

    Google Scholar 

  7. Peters O, Back T, Lindauer U, Busch C, Megow D, Dreier J, et al. Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1998;182:196–205.

    Article  Google Scholar 

  8. Chen XL, Kunsch C. Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr Pharm Des. 2004;108:879–91.

    Article  Google Scholar 

  9. Kobayashi A, Ohta T, Yamamoto M. Unique function of the Nrf2-Keap1 pathway in the inducible expression of antioxidant and detoxifying enzymes. Meth Enzymol. 2004;378:273–86.

    Article  CAS  PubMed  Google Scholar 

  10. Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med. 2004;1011:549–57.

    Article  Google Scholar 

  11. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. Surg Neurol. 2006;663:232–45.

    Article  Google Scholar 

  12. Dubinsky R, Lai SM. Mortality of stroke patients treated with thrombolysis: analysis of nationwide inpatient sample. Neurology. 2006;6611:1742–4.

    Article  Google Scholar 

  13. Sapolsky RM, Steinberg GK. Gene therapy using viral vectors for acute neurologic insults. Neurology. 1999;539:1922–31.

    Google Scholar 

  14. Yenari MA, Dumas TC, Sapolsky RM, Steinberg GK. Gene therapy for treatment of cerebral ischemia using defective herpes simplex viral vectors. Neurol Res. 2001;235:543–52.

    Article  Google Scholar 

  15. Linnik MD, Zahos P, Geschwind MD, Federoff HJ. Expression of bcl-2 from a defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia. Stroke J Cereb Circ. 1995;269:1670–4. discussion 1675.

    Google Scholar 

  16. Hermann DM, Kilic E, Kugler S, Isenmann S, Bahr M. Adenovirus-mediated GDNF and CNTF pretreatment protects against striatal injury following transient middle cerebral artery occlusion in mice. Neurobiol Dis. 2001;84:655–66.

    Article  Google Scholar 

  17. Andsberg G, Kokaia Z, Klein RL, Muzyczka N, Lindvall O, Mandel RJ. Neuropathological and behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats. Neurobiol Dis. 2002;92:187–204.

    Article  Google Scholar 

  18. Greenberg DA, Jin K. From angiogenesis to neuropathology. Nature. 2005;4387070:954–9.

    Article  Google Scholar 

  19. Shen F, Su H, Fan Y, Chen Y, Zhu Y, Liu W, et al. Adeno-associated viral-vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice. Stroke J Cereb Circ. 2006;3710:2601–6.

    Google Scholar 

  20. Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, et al. VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Investig. 2003;11112:1843–51.

    Google Scholar 

  21. Lee RJ, Springer ML, Blanco-Bose WE, Shaw R, Ursell PC, Blau HM. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation. 2000;1028:898–901.

    Google Scholar 

  22. Ozawa CR, Ho JJ, Tsai DJ, Ho DY, Sapolsky RM. Neuroprotective potential of a viral vector system induced by a neurological insult. Proc Natl Acad Sci USA. 2000;9716:9270–5.

    Article  Google Scholar 

  23. Post DE, Van Meir EG. Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells. Gene Ther. 2001;823:1801–7.

    Article  Google Scholar 

  24. Reinblatt M, Pin RH, Bowers WJ, Federoff HJ, Fong Y. Herpes simplex virus amplicon delivery of a hypoxia-inducible soluble vascular endothelial growth factor receptor (sFlk-1) inhibits angiogenesis and tumor growth in pancreatic adenocarcinoma. Ann Surg Oncol. 2005;1212:1025–36.

    Article  Google Scholar 

  25. Qutub AA, Popel AS. A computational model of intracellular oxygen sensing by hypoxia-inducible factor HIF1 alpha. J Cell Sci. 2006;119Pt(16):3467–80.

    Article  Google Scholar 

  26. Yu AY, Frid MG, Shimoda LA, Wiener CM, Stenmark K, Semenza GL. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. Am J Physiol. 1998;2754(Pt 1):L818–26.

    Google Scholar 

  27. Brooke SM, Bliss TM, Franklin LR, Sapolsky RM. Quantification of neuron survival in monolayer cultures using an enzyme-linked immunosorbent assay approach, rather than by cell counting. Neurosci Lett. 1999;2671:21–4.

    Article  Google Scholar 

  28. Ho DY, Fink SL, Lawrence MS, Meier TJ, Saydam TC, Dash R, et al. Herpes simplex virus vector system: analysis of its in vivo and in vitro cytopathic effects. J Neurosci Meth. 1995;572:205–15.

    Article  Google Scholar 

  29. Leonard MO, Kieran NE, Howell K, Burne MJ, Varadarajan R, Dhakshinamoorthy S, et al. Reoxygenation-specific activation of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia-reperfusion injury. FASEB J. 2006;2014:2624–6.

    Article  Google Scholar 

  30. Shih AY, Imbeault S, Barakauskas V, Erb H, Jiang L, Li P, et al. Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo. J Biol Chem. 2005;28024:22925–36.

    Article  Google Scholar 

  31. Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, et al. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci. 2003;238:3394–406.

    Google Scholar 

  32. Shih AY, Li P, Murphy TH. A small-molecule-inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo. J Neurosci. 2005;2544:10321–35.

    Article  Google Scholar 

  33. Sun X, Erb H, Murphy TH. Coordinate regulation of glutathione metabolism in astrocytes by Nrf2. Biochem Biophys Res Commun. 2005;3262:371–7.

    Article  Google Scholar 

  34. Lee JY, Lee YS, Kim JM, Kim KL, Lee JS, Jang HS, et al. A novel chimeric promoter that is highly responsive to hypoxia and metals. Gene Ther. 2006;1310:857–68.

    Google Scholar 

  35. Hurttila H, Koponen JK, Kansanen E, Jyrkkanen HK, Kivela A, Kylatie R, et al. Oxidative stress-inducible lentiviral vectors for gene therapy. Gene Ther. 2008;1518:1271–9.

    Article  Google Scholar 

  36. Semenza GL, Agani F, Feldser D, Iyer N, Kotch L, Laughner E, et al. Hypoxia, HIF-1, and the pathophysiology of common human diseases. Adv Exp Med Biol. 2000;475:123–30.

    Article  CAS  PubMed  Google Scholar 

  37. Shih AY, Erb H, Murphy TH. Dopamine activates Nrf2-regulated neuroprotective pathways in astrocytes and meningeal cells. J Neurochem. 2007;1011:109–19.

    Google Scholar 

  38. Siebert A, Desai V, Chandrasekaran K, Fiskum G, Jafri MS. Nrf2 activators provide neuroprotection against 6-hydroxydopamine toxicity in rat organotypic nigrostriatal cocultures. J Neurosci Res. 2009;877:1659–69.

    Article  Google Scholar 

  39. Shah ZA, Li RC, Thimmulappa RK, Kensler TW, Yamamoto M, Biswal S, et al. Role of reactive oxygen species in modulation of Nrf2 following ischemic reperfusion injury. Neuroscience. 2007;1471:53–9.

    Article  Google Scholar 

  40. Fink DJ, DeLuca NA, Goins WF, Glorioso JC. Gene transfer to neurons using herpes simplex virus-based vectors. Annu Rev Neurosci. 1996;19:265–87.

    Article  CAS  PubMed  Google Scholar 

  41. Glorioso JC, Goins WF, Fink DJ, DeLuca NA. Herpes simplex virus vectors and gene transfer to brain. Dev Biol Stand. 1994;82:79–87.

    CAS  PubMed  Google Scholar 

  42. Bergeron M, Yu AY, Solway KE, Semenza GL, Sharp FR. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci. 1999;1112:4159–70.

    Article  Google Scholar 

  43. Ozaki H, Yu AY, Della N, Ozaki K, Luna JD, Yamada H, et al. Hypoxia inducible factor-1alpha is increased in ischemic retina: temporal and spatial correlation with VEGF expression. Investig Ophthalmol Vis Sci. 1999;401:182–9.

    Google Scholar 

  44. Dumas TC, Sapolsky RM. Gene therapy against neurological insults: sparing neurons versus sparing function. Trends Neurosci. 2001;2412:695–700.

    Article  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. Jewed Alam and Dr. Howard Federoff for kindly providing the pEF-Nrf2 plasmid and the replication defective HSV plasmid, respectively. This work was supported by the NIH-Center for Cerebrovascular Disease no. 2P01 NS37520. MYC was supported by the American Heart Association (no. 0525025Y).

Author information

Authors and Affiliations

  1. Department of Biology, Stanford University, 371 Serra Mall, Stanford, CA, 94305-5020, USA

    Michelle Y. Cheng, I-Ping Lee, Michael Jin & Robert M. Sapolsky

  2. Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive, Rm 281A, Stanford, CA, 94305-5327, USA

    Michelle Y. Cheng, Guohua Sun, Heng Zhao, Gary K. Steinberg & Robert M. Sapolsky

Authors
  1. Michelle Y. Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I-Ping Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guohua Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heng Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gary K. Steinberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert M. Sapolsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michelle Y. Cheng.

Additional information

Michelle Y. Cheng and I-Ping Lee contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cheng, M.Y., Lee, IP., Jin, M. et al. An Insult-Inducible Vector System Activated by Hypoxia and Oxidative Stress for Neuronal Gene Therapy. Transl. Stroke Res. 2, 92–100 (2011). https://doi.org/10.1007/s12975-010-0060-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12975-010-0060-2

Keywords

Search

Navigation