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The Hammerhead Ribozyme Mediated Repression of VEGF for Cancer Gene Therapy
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Abstract:
Objective: To establish a novel and robust hammerhead ribozyme system against the Vascular Endothelial Growth Factor (VEGF) for anti-cancer gene therapy. Methods： The structural analysis of the 2ndstructure of the VEGF RNA and the vector construction of hammerhead ribozymes (1-4) against VEGF leader region(+1 to+75); The in vitro analyses of ribozyme mediated specific cleavage; The in cell evaluation of the ribozyme mediated cleavage of the VEGF RNA. Results：Ribozymes targeting +8, +36, or +71 (Rz1,3 and 4) of the exposed region or +17 (Rz2) of the unexposed region of VEGF RNA were constructed in pGVal and pFB retroviral vector systems; Rz1,3 and 4, but not Rz2, specifically cleaved the VEGF RNA and brought the VEGF RNA level down to 61.7%, 27.6% and 44.8%(luciferase activity)as well as 66.3%, 27.0% and 30.0%(protein level) of the control; The same set of ribozymes reduced the co-transfected VEGF-LUC RNA level down to 81.4%，56.6% and 69.1% of the control in a transient transfection analysis and essentially abolished the endogenous VEGF RNA in the stable transfected setting. Conclusion: We have established three effective hammerhead ribozyme vector systems targeting +8, +36 and +71 of the VEGF RNA.
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