Engineered Thymidine-Active Deoxycytidine Kinase for Bystander Killing of Malignant Cells. Methods Mol Biol 2019;1895:149-163
Date
12/13/2018Pubmed ID
30539536DOI
10.1007/978-1-4939-8922-5_12Scopus ID
2-s2.0-85058602227 (requires institutional sign-in at Scopus site)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.
Author List
Neschadim A, Medin JAAuthor
Jeffrey A. Medin PhD Professor in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsApoptosis
Bromodeoxyuridine
Bystander Effect
Cell Line, Tumor
Deoxycytidine Kinase
Genes, Transgenic, Suicide
Genetic Therapy
HEK293 Cells
Humans
Male
Mice
Mice, SCID
Neoplasms
Prodrugs
Thymidine
