Team:Slovenia/SafetyMechanismsTermination
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<p><b>Figure 1. Mechanism of action of the HSV-TK/GCV system.</b></p> | <p><b>Figure 1. Mechanism of action of the HSV-TK/GCV system.</b></p> | ||
<p>Following the BioBrick Standard Assembly technique we inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK:TK30, (BBa_K404113)) under the control of the CMV promoter (Figure 2). HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999).</p> | <p>Following the BioBrick Standard Assembly technique we inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK:TK30, (BBa_K404113)) under the control of the CMV promoter (Figure 2). HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999).</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/0/05/Svn12_safety_mechanisms_termination_fig2.png"></img> | ||
+ | <p><b>Figure 2. Assembly of mGMK:TK30 (BBa_K404113) under the control of CMV promoter.</b> Insert (mGMK:TK30, (BBa_K404113)) was digested with EcoRI and SpeI and pcDNA3 vector with EcoRI and XbaI, loaded on an agarose gel and the appropriate bands were purified. Vector and insert were ligated and transformed into <i>E. coli</i> strain DH5α. The DNA sequence of the resulting plasmid pPCMV_mGMK:TK30 was confirmed by DNA sequencing.</p> | ||
+ | <p>We transfected HEK293 cells with the pPCMV_mGMK:TK30 plasmid and monitored cell survival after the addition of ganciclovir. We daily observed cells with an light microscope and monitored the morphological signs of decreased viability. Additionally we stained cells with Hoechst and SytoxGreen514 dye to distinguish between live and dead cells with the help of a confocal microscope. We quantified viable cell numbers at different time points by cell counting and flow cytometry. For detailed protocols please refer to the "Experimental methods" section.</p> | ||
+ | </p> | ||
+ | <h2>Results</h2> | ||
+ | |||
<h2 style="color:grey;">References</h2> | <h2 style="color:grey;">References</h2> |
Revision as of 17:09, 25 September 2012
Termination
In order to terminate our system at the end of the therapy, we designed our therapeutic cells so that they would produce thymidine kinase.
Expression of thymidine kinase is not deleterious to cell growth.
The addition of the prodrug ganciclovir, which is converted into a toxic compound in cells producing thymidine kinase, efficiently initiates the apoptosis of therapeutic cells, making this mechanism virtualy free of leaky apoptosis.
The Thymidine kinase/Ganciclovir system in detail
Suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT) is widely used in cancer treatment. One of the most used GDEPT systems is the herpes simplex virus thymidine kinase (HSV-TK) with purine nucleoside analog ganciclovir (GCV) as a prodrug. Systemic administration of the prodrug ganciclovir induces apoptosis only in cells transfected with HSV-thymidine kinase while the untransfected cells survive. Unlike human thymidine kinase, HSV-thymidine kinase is able to phosphorylate ganciclovir to form ganciclovir-monophosphate, which is then phosphorylated to ganciclovir-diphosphate followed by ganciclovir-triphosphate. Ganciclovir-triphosphate is then incorporated into the DNA, which causes inhibition of DNA synthesis and subsequently leads to apoptosis (Ardiani et al., 2010). We used the HSV-thymidine kinase fused to mouse guanylate kinase (mGMK:TK30, BioBrick BBa_K404113, prepared by the Freiburg_Bioware team in 2010) in our system because it improves phosphorylation of ganciclovir-monophosphate to ganciclovir-diphosphate, thus preventing accumulation of ineffective intermediate products (i.e. GCV-MP, GCV-DP) due to the limited ability of the endogenous guanylate kinase (Figure 1).
We successfully implemented HSV-TK/GCV system to our cellular device to function as a controllable “safety switch”. This means that we can inactivate our cellular device whenever we want, after or even in the middle of therapy, simply by administering ganciclovir to the patient.
![](https://static.igem.org/mediawiki/2012/8/83/Svn12_safety_mechanisms_termination_fig1.png)
Figure 1. Mechanism of action of the HSV-TK/GCV system.
Following the BioBrick Standard Assembly technique we inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK:TK30, (BBa_K404113)) under the control of the CMV promoter (Figure 2). HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999).
![](https://static.igem.org/mediawiki/2012/0/05/Svn12_safety_mechanisms_termination_fig2.png)
Figure 2. Assembly of mGMK:TK30 (BBa_K404113) under the control of CMV promoter. Insert (mGMK:TK30, (BBa_K404113)) was digested with EcoRI and SpeI and pcDNA3 vector with EcoRI and XbaI, loaded on an agarose gel and the appropriate bands were purified. Vector and insert were ligated and transformed into E. coli strain DH5α. The DNA sequence of the resulting plasmid pPCMV_mGMK:TK30 was confirmed by DNA sequencing.
We transfected HEK293 cells with the pPCMV_mGMK:TK30 plasmid and monitored cell survival after the addition of ganciclovir. We daily observed cells with an light microscope and monitored the morphological signs of decreased viability. Additionally we stained cells with Hoechst and SytoxGreen514 dye to distinguish between live and dead cells with the help of a confocal microscope. We quantified viable cell numbers at different time points by cell counting and flow cytometry. For detailed protocols please refer to the "Experimental methods" section.
Results
References
Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L., Spies, T. (1999) Activation of NK Cells and T Cells by NKG2D, a receptor for Stress-Inducible MICA. Science 285, 727-729.
Borrego, F., Kabat, J., Kim, D.K., Lieto, L., Maasho, K., Peña, J., Solana, R., Coligan J.E. (2001) Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol. Immunol. 38, 637-660.
Groh, V., Rhinehart, R., Secrist, H., Bauer., S., Grabstein, K.H., Spies, T. (1999) Broad tumor-associated expression and recognition by tumor-derived gd T cells of MICA and MICB. Proc. Natl. Acad. Sci. 96, 6879–6884.
Salih, H.R., Rammensee, H.G., Steinle, A. (2002) Cutting Edge: Down-Regulation of MICA on Human Tumors by Proteolytic Shedding. J Immunol. 169, 4098-4102.
Stenile, A., Li, P., Morris, D.L., Groh, V., Lanier, L.L., Strong, R.K., Spies, T. (2001) Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family. Immunogenet. 53, 279-287.