A preliminary step of a novel strategy in suicide gene therapy with lentiviral vector

• Main Authors: Jahan Afrooz Ghanbari, Mansoor Salehi, Arezoo Karam Zadeh, Sedigheh Momen Zadeh, Vahid Bahram Beigi, Hossein Khan Ahmad, Behzad Mahaki, Mina Beiraghdar
• Format: Article
• Language: English
• Published: Wolters Kluwer Medknow Publications 2014-01-01
• Series: Advanced Biomedical Research
• Subjects: Apoptosis-inducing genes; CMV promoter; gene therapy; GFP; lentiviral vectors; RΔU3 sequence
• Online Access: http://www.advbiores.net/article.asp?issn=2277-9175;year=2014;volume=3;issue=1;spage=7;epage=7;aulast=Ghanbari
• ISSN: 2277-9175; 2277-9175

Background: One of the challenges in lentiviral vector-based suicide gene therapy by toxin or apoptosis-inducing genes is death of packaging cells. Therefore, the process of production of these lentiviral particles would be stopped in this step. We proposed that insertion of a reverse promoter between R and U5 regions of 5′ long terminal repeat (LTR) in transfer plasmid could be considered as a solution for this problem. But it is not known, whether the insertion of RΔU3 sequence between the promoter and target gene in proviral genome during the life-cycle of lentivirus may interfere whit gene expression in target cells. Materials and Methods: The following were performed in this study: insertion of RΔU3 sequence in pEGFP-N1 plasmid, evaluation of the expression of eGFP gene after calcium phosphate co-precipitation transfection of pCMV-RΔU3-GFP construction in 293T cells, and quantitative assay of eGFP gene by flow cytometry technique. Results: Our results from flow cytometry technique analysis showed that there was no significant difference between the expression of eGFP gene in transfected cells with pEGFP-N1 and pCMV-RΔU3-GFP plasmids (P > 0.05). Conclusion: In this step of our strategy, we demonstrated that modification of orientation and location of promoter may overcome some issues in lentiviral suicide gene therapy, especially when toxin or apoptosis-inducing genes are used.