Contents 1 The Kill Switch as a Safety Lock 1.1 The Kill Switch in Detail: The Ganciclovir /Thymidine Kinase System 1.2 The TK/GCV System: Implementation 1.3 The Alternative System: The 5-Fluorocytosine/ Cytosine Deaminase System 1.4 References

The Kill Switch as a Safety Lock

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The Kill Switch in Detail: The Ganciclovir /Thymidine Kinase System

A rapidly evolving research area in the field of oncology is the one focused on bacterial based cancer therapies. In this type of therapies, bacteria like E. Coli and Salmonella typhimurium are used as tumor targeting systems. These bacteria are used to deliver cytotoxic agents, immunostimulatory cytokine, and genetic material to enable host-cell production of these molecules, and induce cell death in this way. forbesBactN.S. Forbes, Engineering the perfect(bacterial) cancer therapy. Nature , 785-794 (2010) Using the bacteria for delivering genetic material to the tumor cells is similar to the suicide gene therapy, also known as gene-directed enzyme prodrug therapy (GDEPT), which is widely used in cancer treatment. BugajNextGenTherL.J. Bugaj, D.V. Schaffer, Bringing next-generation therapeutics to the clinic through synthetic biology. Current Opinion in Chemical Biology 16, 355-361 (2012) One of the best characterized and used GDEPT systems is the herpes simplex virus thymidine kinase (HSV-TK) enzyme with purine nucleoside analog ganciclovir (GCV) as a prodrug. Systemic administration of the prodrug ganciclovir induces apoptosis only in the cells that express the HSV-thymidine kinase enzyme, sparing the life of the cells that do not express this enzyme. aghiEtAlGCVFluM. Aghi, C.M. Kramm, T.C. Chou, X.O. Breakefield, E.A. Chiocca, Synergistic Anticancer Effects of Ganciclovir/Thymidine Kinase and 5-Fluorocytosine/Cytosine Deaminase Gene Therapies. Journal of the National Cancer Institute 90, 370-380 (1998) Figure 1 depicts the complete process of cell death induced by the HSV-TK/GCV system.

We propose to use this system as our killing mechanism, considering that it is well characterized and used in applications related to the principle of our device. However at this point of our project the in vitro implementation of this system is not possible and thus we focused on a pharmacokinetics model of the prodrug distribution in the body and the elimination of the CEST protein producing bacteria from the system. The following section describes the the main results from the pharmacokinetics model.

The TK/GCV System: Implementation

We developed a pharmacokinetics model of the prodrug Ganciclovir distribution and the systemic elimination of the CEST protein production device. The complete results can be found in the Drylab section of our wiki, but here we present a summary of the most important conclusions.

• Elimination rate
• Ganciclovir distribution

The Alternative System: The 5-Fluorocytosine/ Cytosine Deaminase System

Similar to the GCV/HSV-TK GDEPT system, is the Escherichia coli cytosine deaminase (CD) enzyme with prodrug 5-fluorocytosine. Like the prodrug ganciclovir, systemic administration of the prodrug 5-fluorocytosine induces apoptosis only in the cells that express the cytosine deaminase enzyme. Cell death is induced when the CD deaminates the prodrug 5-fluorocytosine, forming 5-fluorouracil, which is further metabolized into FdUMP. FdUMP inhibits the enzyme thymidylate synthetase which converts dUMP into thymidylate. Because thymidylate can only be produced de novo by the enzyme thymidylate synthetase, the inhibition of the enzyme eventually generates a deficiency of dTTP, leading to cell death. aghiEtAlGCVFlu Figure 2 shows a detailed graphic explanation of the complete process.

Given that the chasis of our device is E. coli, the implementation of the CD/5’fluorocytosine system on our device could be an alternative to the GCV/HSV-TK system. We didn’t explore this alternative further but we will consider it when testing the kill switch in vitro in case the GCV/HSV-TK is not successful in inducing apoptosis in our CEST protein producing bacteria.

References