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

Single Cell Analysis: Flourescence Microscopy

Since their advent, proteomics and genomics have developed in ways that underline the rate and volume of data acquisition and analysis. They have, by necessity, worked on large populations of cells and thereby reported on population averages rather than their distributions.In this process they have missed rare, but important events and been unable to analyse cells that are only produced in small numbers. This is of course not by choice but owing to a paucity of techniques that allow experimentalists to measure protein levels at the single-cell level.

In addition, genome sequence information provides powerful insights into cellular complexity but limited information pertaining to how individual parts of a cell are work together in time and space to form dynamic cellular processes and how cellular interactions consequently translate to create higher order functions. This currently hampers an understanding of the mechanisms behind the morphological design of organisms that depends on programmes of cellular division, apoptosis, immune response etc. that are closely linked to these spatially and temporally dependent signals.

Parameters based upon averages of large populations are often misleading. Cellular heterogeneity is widespread in bacteria and increasingly apparent in eukaryotic cells. The complex and highly interconnected network of signalling pathways, their spatially dependent nature and reliance upon low-abundance molecules produces stochastic behaviour that subsequently underpins heterogeneity in cellular systems. The noise in biological function subsequently expresses itself in many different forms, from noise-driven divergence of individual cell fates through to noise-induced amplification of signals.

Similarly, the study of rare cells such as stem cells and progenitor cells does not lend itself to high throughput population-based protocols. In these cases, the development of single-cell analysis techniques that allow multiple measurements to be conducted on the same cell as a function of time is vital if we are to unravel the inner workings of these extraordinary systems.

Focusing on the characteristics of a group can obscure the differences between the individuals in it. Yet when it comes to biological cells, scientists typically derive information about their behaviour, status, and health from the collective activity of thousands or millions of them. Analyzing individual cells allows researchers to distinguish between a uniform population of cells and a group of cells with members having, say, different protein content.

A more precise understanding of differences between individual cells could lead to better treatments for diseases such as cancer, diabetes etc. Detecting minute differences between individual cells could improve medical tests and treatments. An understanding of the relationship between biological heterogeneity and signalling pathway regulation that may result in disease states is therefore critical and offers the potential to drive novel therapeutic interventions developed in response to single-cell behaviours.

Thus, Single-cell studies are crucial in order to productively study the complexity of intracellular processes.

However, tools that are capable of harvesting large amounts of proteomic data from single cells remain rather limited, largely owing to the difficulty involved in dealing with the small volumes and quantities of analytes concerned. Despite the limitations, over the last decade or two, there has been significant progress in developing assays capable of determining levels of specific proteins and interrogating enzyme activity in single cells .Fluorescence Microscopy is one such method which can be sucessfully used for Single cell analysis.

Lots of agents are used for the fluorescence, one such widely used protein is EGFP (Enhanced Green Fluorescent Protein). In the 1960s and 1970s, GFP, was first purified from Aequorea victoria and its properties studied by Osamu Shimomura. In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca2+ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green. However, it’s utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene. A 37 °C folding efficiency (F64L) point mutant to this scaffold yields enhanced GFP (EGFP) which was discovered in 1995 by the lab of Ole Thastrup. EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M−1cm−1. The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M−1cm−1.

EGFP expressing cells under Flourescence Microscope

EGFP can also be expressed in different structures enabling morphological distinction. In our cases, the gene for the production of EGFP is spliced into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that is, the gene's regulatory sequence now controls the production of EGFP, in addition to the tagged protein. In cells where the gene is expressed, and the tagged proteins are produced, EGFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. The fluorescence can be used to even identify position of the target protein and the its amount produced at a specific time point.

Analysis of such time lapse movies has redefined the understanding of many biological processes in our case helped us to understand protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material.