Abstract
BackgroundRegulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.ResultsWhen tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)2R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.ConclusionsThis suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.
Highlights
Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science
Results truncated Bid (tBid) induces efficiently and rapidly cell death in HeLa SS6 cells To estimate the susceptibility of the cell line HeLa SS6 towards induction of cell death by the human proapoptotic protein tBid, we transiently co-transfected these cells with the plasmid pCMV-tBid which constitutively expresses tBid under control of a CMV-promoter, and with pEGFP-C1 allowing us to determine transfection efficiencies by measuring green fluorescence. 24 hours after transfection, GFP was expressed in 63% of the HeLa SS6 cells (Figure 1A)
Induction of cell death by tBid in the suicide vector pLtBid(INS)2R strongly depends on the presence of both human immunodeficiency virus type 1 (HIV-1) Tat and Rev First, we characterized the control vector pLRed(INS)2R (Figure 1B) in transiently transfected HeLa SS6 cells or in HeLa-Tat cells, constitutively expressing the HIV-1 Tat protein
Summary
Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection. Both basic and clinical science can greatly benefit from vectors that induce cell death in a temporally and spatially controlled manner. Transient or inducible expression of tBid leads to rapid and efficient induction of apoptosis in a variety of cell lines [23,24,25]
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