Team:Slovenia/SafetyMechanismsTermination

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<h3>The Thymidine kinase/Ganciclovir system in detail</h3>
<h3>The Thymidine kinase/Ganciclovir system in detail</h3>
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<p>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 <b>herpes simplex virus thymidine kinase (HSV-TK)</b> with <b>purine nucleoside analog ganciclovir (GCV)</b> 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>i.e.</i> GCV-MP, GCV-DP) due to the limited ability of the endogenous guanylate kinase (Figure 1).</p>
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<p>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 <b>herpes simplex virus thymidine kinase (HSV-TK)</b> with <b>purine nucleoside analog ganciclovir (GCV)</b> 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).</p>
<p>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.</p>
<p>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.</p>

Revision as of 16:53, 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.

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.