HIV-specific promoters to eliminate HIV-infected cells by gene therapy
Nejat Düzgüneş: 0000-0001-6159-1391
41th Annual Meeting & Exhibition of the American Association for Dental Research (AADR)
March 21-24, 2012
Date of Presentation
Journal of Dental Research
Journal Volume Number
91 (Special issue A
Objectives: Current therapies against HIV infection are unable to eradicate the chromosomally integrated proviral genome. Successful therapy of HIV/AIDS requires a method to specifically kill HIV-infected cells. We are developing HIV-specific promoters to drive the expression of suicide genes that will kill HIV-infected cells, but not uninfected cells. Here we examined the expression of a reporter gene, luciferase, driven by the HIV promoter, LTR, and its mutants. Our aim is to design and synthesize a promoter that responds to the HIV transcriptional activator, Tat, but not to cellular transcription factors. Methods: The full-length LTR and five progressively truncated versions of the promoter were generated using PCR-based cloning techniques (Bionexus), and designated LTR1ÐLTR6. These promoters were inserted into the pGL3 Basic Vector (Promega) encoding luciferase. These plasmids were transfected into HeLa cells, and HeLa-tat-III cells that constitutively express Tat, using the cationic liposome, Metafectene (Biontex). Luciferase activity (relative light units (RLU)/ml cell lysate) was measured 48 h later, using the Luciferase Assay System (Promega). Results: Luciferase expression from the LTR2 construct increased from 1,060 RLU/ml in HeLa cells to 108,187 RLU/ml in HeLa-tat-III cells, a 102-fold increase. In LTR2, the LTR modulatory region is truncated, but the NF-?B binding region is maintained. Luciferase expression from LTR1 containing the wild type HIV promoter, was 595 RLU/ml in HeLa cells, and 30,373 in HeLa-tat-III cells. In LTR3 without the NF-?B binding region the corresponding values were 498 and 25,387 RLU/ml. The other constructs resulted in much lower gene expression. Conclusions: HIV-specific cell killing may be possible by generating a suicide gene construct driven by the LTR2 promoter. It is expected that the incorporation of this, or a more Tat-specific construct, into a lentiviral vector may lead to the therapeutic transduction of all HIV-harboring cells. Supported by Research Awards 03-Activity-071 and 03-Activity-076.
HIV-specific promoters to eliminate HIV-infected cells by gene therapy.
Paper presented at 41th Annual Meeting & Exhibition of the American Association for Dental Research (AADR) in Tampa, FL.