Suicide Gene Therapy against HIV :Thymidine Kinase gene

Acquired immunodeficiency syndrome (AIDS) and its associated disorders are caused by human immunodeficiency virus (HIV). HIV exhibits a tropism for CD4+ T lymphocytes, which constitute the primary target for HIV infection in vivo. Initial infections appear to be latent and are characterized by an extended, asymptomatic stage of pathogenesis. Disease progression results in a state of prolific viral replication that eventually leads to the massive depletion of CD4+ T lymphocytes that occurs during AIDS.  Even though substantial progress has been made in the molecular characterization of HIV, therapeutic treatment of HIV mediated pathogenesis has proved difficult to attain, particularly due to uncertainties in understanding mechanisms related to the persistence of viral latency.

Resting T lymphocytes are nonpermissive for HIV replication; even then the virus efficiently binds to the CD4 receptor and is internalized.  From this latent state, the virus can be initiated into productive infection by factors that activate quiescent CD4+ lymphocytes into cellular proliferation. It appears that the latent provirus is activated by the same inducible cellular transcription factors that promote T-cell proliferation upon presentation of the appropriate antigen. This feature of HIV replication raises therapeutic possibilities; agents that are toxic to activated HIV-infected T cells would be expected to inhibit HIV replication and the ensuing pathogenesis. 

HIV-infected T cells upon activation, initiate a program of viral gene expression that is stringently controlled by two nuclear regulatory proteins coding viral genes tat and rev. The main function of tat gene product, Tat, is transcriptional activation from the viral 5' long terminal repeat (LTR) promoter by binding to structured RNA target sequence, the transactivation response element (TAR). The action of the rev gene product, Rev, is posttranscriptional; Rev selectively induces the nuclear export of a constitutively expressed pool of structural-gene mRNAs that contain a cis-acting sequence of extensive secondary structure, the Rev-responsive element (RRE). The viral cis-acting sequences TAR and RRE, whose regulatory activity is tightly controlled by Tat and Rev, respectively, afford an excellent means for the targeted expression of cytotoxic agents in HIV-infected cells that express these regulatory proteins.
 
Suicide Gene therapy is a technique for modifying the cellular genome for rendering cells sensitive to chemotherapeutics or toxins by introducing “suicide genes”. Here conditionally cytotoxic human herpes virus type- 6 thymidine kinase gene (HHV-6 TK) is used as suicide gene in Tat expressing HIV infected cells.  HSV-1 TK expression is not deleterious to mammalian cells, but it can, unlike mammalian thymidine kinase, selectively phosphorylate certain nucleoside analogs such as acyclovir and ganciclovir (GCV) to their monophosphate..  Ganciclovir(GCV),  is phosphorylated first by the viral thymidine kinase to nucleoside monophosphate (GCV-MP) and then by cell kinases to yield the triphospho form of the drug(GCV-TP).Gancyclovir triphosphate  when  incorporated into DNA, leads to inhibition of DNA synthesis .This TK-GCV system induces accumulation of p53 and increases cell surface expression of death receptors  leading to apoptosis involving the Fas-associated death domain protein (FADD) and caspases. One more advantage of the HSV-TK-GCV system is the bystander killing effect whereby HSV-TK positive cells exposed to GCV are lethal to surrounding HSV-TK negative cells via transport of GCV-MP, GCV-BP, and GCV-TP trough gap junctions to adjacent cells.Thus using HIV-2 LTR as inducible promoter for the thymidine kinase gene ,the HIV infected cells can be specifically killed .Similar strategy were  used earlier for specific killing of  HIV infected cells  where  LTR promoter is used for selective expression of  pro-apoptotic Bax gene leading to apoptotic cell death of tat expressing cells ( McCoubrie JE. et al,2004) or  LTR- herpes simplex virus (HSV) virion host shutoff gene (vhs) construct where vhs  encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis in HIV infected cells ( Hamouda T. et al,1997).

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Suicide Gene Therapy against HIV : Thymidine Kinase gene