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Engineered Thymidine-Active Deoxycytidine Kinase for Bystander Killing of Malignant Cells

  • Anton Neschadim
  • Jeffrey A. MedinEmail author
Protocol
  • 671 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1895)

Abstract

Suicide transgenes encode proteins that are either capable of activating specific prodrugs into cytotoxic antimetabolites that can trigger cancer cell apoptosis or are capable of directly inducing apoptosis. Suicide gene therapy of cancer (SGTC) involves the targeted or localized delivery of suicide transgene sequences into tumor cells by means of various gene delivery vehicles. SGTC that operates via the potentiation of small-molecule pharmacologic agents can elicit the elimination of cancer cells within a tumor beyond only those cells successfully transduced. Such “bystander effects”, typically mediated by the spread of activated cytotoxic antimetabolites from the transduced cells expressing the suicide transgene to adjacent cells in the tumor, can lead to a significant reduction of the tumor mass without the requirement of transduction of a high percentage of cells within the tumor. The spread of activated cytotoxic molecules to adjacent cells is mediated primarily by diffusion and normally involves gap junctional intercellular communications (GJIC). We have developed a novel SGTC system based on viral vector-mediated delivery of an engineered variant of human deoxycytidine kinase (dCK), which is capable of phosphorylating uridine- and thymidine-based nucleoside analogues that are not substrates for wild-type dCK, such as bromovinyl deoxyuridine (BVdU) and L-deoxythymidine (LdT). Since our dCK-based SGTC system is capable of mediating strong bystander cell killing, it holds promise for clinical translation. In this chapter, we detail the key procedures for the preparation of recombinant lentivectors for the delivery of engineered dCK, transduction of tumor cells, and evaluation of bystander cell killing effects in vitro and in vivo.

Key words

Suicide gene therapy (SGT) Cancer Tumor xenografts Deoxycytidine kinase (dCK) Bromovinyl deoxyuridine (BVdU) l-Deoxythymidine (LdT) 

Notes

Acknowledgments

A.N. was funded by the Canadian Institutes of Health Research (CIHR) Training Program in Regenerative Medicine. J.A.M. is funded by the Midwest Athletes against Childhood Cancer (MACC) Fund.

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centre for Innovation, Canadian Blood ServicesTorontoCanada
  2. 2.Departments of Pediatrics and BiochemistryMedical College of WisconsinMilwaukeeUSA

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