Faculty Collaboration Database

Evaluation of Bystander Cell Killing Effects in Suicide Gene Therapy of Cancer: Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis. Methods Mol Biol 2015;1317:55-67

Date

06/15/2015

Pubmed ID

26072401

DOI

10.1007/978-1-4939-2727-2_4

Scopus ID

2-s2.0-84931265253 (requires institutional sign-in at Scopus site)   3 Citations

Abstract

Suicide gene therapy of cancer (SGTC) entails the introduction of a cDNA sequence into tumor cells whose polypeptide product is capable of either directly activating apoptotic pathways itself or facilitating the activation of pharmacologic agents that do so. The latter class of SGTC approaches is of the greater utility in cancer therapy owing to the ability of some small, activated cytotoxic compounds to diffuse from their site of activation into neighboring malignant cells, where they can also mediate destruction. This phenomenon, termed "bystander killing", can be highly advantageous in driving significant tumor regression in vivo without the requirement of transduction of each and every tumor cell with the suicide gene. We have developed a robust suicide gene therapy enzyme/prodrug system based on an engineered variant of the human thymidylate kinase (TMPK), which has been endowed with the ability to drive azidothymidine (AZT) activation. Delivery of this suicide gene sequence into tumors by means of recombinant lentivirus-mediated transduction embodies an SGTC strategy that successfully employs bystander cell killing as a mechanism to achieve significant ablation of solid tumors in vivo. Thus, this engineered TMPK/AZT suicide gene therapy axis holds great promise for clinical application in the treatment of inoperable solid tumors in the neoadjuvant setting. Here we present detailed procedures for the preparation of recombinant TMPK-based lentivirus, transduction of target cells, and various approaches for the evaluation of bystander cell killing effects in SGCT in both in vitro and in vivo models.

Author List

Sato T, Neschadim A, Nakagawa R, Yanagisawa T, Medin JA

Author

Jeffrey A. Medin PhD Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Bystander Effect
Cell Death
Cell Line, Tumor
Cell Proliferation
Colorimetry
Gene Expression
Genes, Transgenic, Suicide
Genetic Engineering
Genetic Therapy
HEK293 Cells
Humans
Lentivirus
Male
Mice, Inbred NOD
Mice, SCID
Neoplasms
Nucleoside-Phosphate Kinase
Prodrugs
Recombinant Proteins
Transduction, Genetic
Transgenes
Zidovudine