Team:TECHNION-ISRAEL/kill switch
Ganciclovir Kill Switch
In light of our delay mechanism epitopes are displayed on the cellular membrane only after engraftment within the bone marrow. Unfortunately no biologically inducible system is perfect and there will always be a basal level of “leakiness”. Additionally, bone marrow cells routinely migrate to the bloodstream. For this reason, as well as ethical considerations, we have implemented a "Kill switch" based on the use of the Ganciclovir prodrug and transfection of the Herpes Simplex Virus – Thymidine Kinase (HSV-TK).
Transfection of HSV-TK, followed by treatment with the prodrug ganciclovir (GCV), is one of the most commonly used [1] experimental and clinical methods for Gene-Directed Enzyme Prodrug Therapy (GDEPT). The concept of GDEPT has existed for over 20 years, but its clinical importance has grown exponentially in the past 5 years with more and more GDEPT based systems entering clinical trials. GDEPT systems are comprised of three components: An inactive drug (“prodrug”), a gene coding for an enzyme that converts the inactive prodrug to an active drug, and a delivery mechanism. Ideally, the prodrug should be non-toxic or minimally toxic prior to activation by enzyme but highly toxic after enzymatic activation [1] .
As shown below expression of HSV-TK gene leads to the synthesis of viral thymidine kinases. These kinases convert GCV into GCV monophosphate which is subsequently converted into a toxic triphosphate form by natural cellular kinases. GCV triphosphate is analogous to 2'-deoxyguanosine triphosphate (dGTP) and therefore incorporates into replicating DNA causing premature strand termination, replication failure and apoptosis (Figure 1) [2] .
Figure 1: The Ganciclovir System
Our goal is to add an additional cistron, which will code for HSV-TK, to our display mechanism. Accordingly, transfected HSCs expressing epitope will concurrently express HSV-TK. In the case of an allergic reaction, or patient dissatisfaction, treatment with Ganciclovir will induce apoptosis in all epitope expressing HSCs.
- Niculescu-Duvaz, Ion, et al. "Gene-directed enzyme prodrug therapy." Bioconjugate chemistry 9.1 (1998): 4-22.
- Zhang, Jin, Vijay Kale, and Mingnan Chen. "Gene-directed enzyme prodrug therapy." The AAPS journal 17.1 (2015): 102-110.
Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel
igem.technion.2017@gmail.com
