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.
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References
Sato T, Neschadim A, Lavie A et al (2013) The engineered thymidylate kinase (TMPK)/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer. PLoS One 8(10):e78711. https://doi.org/10.1371/journal.pone.0078711
Freeman SM, Abboud CN, Whartenby KA et al (1993) The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 53(21):5274–5283
Fick J, Barker FG 2nd, Dazin P et al (1995) The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro. Proc Natl Acad Sci U S A 92(24):11071–11075
Mesnil M, Yamasaki H (2000) Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication. Cancer Res 60(15):3989–3999
Colombo F, Barzon L, Franchin E et al (2005) Combined HSV-TK/IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results. Cancer Gene Ther 12(10):835–848. https://doi.org/10.1038/sj.cgt.7700851
Hasenburg A, Tong XW, Rojas-Martinez A et al (1999) Thymidine kinase (TK) gene therapy of solid tumors: valacyclovir facilitates outpatient treatment. Anticancer Res 19(3B):2163–2165
Aguilar LK, Shirley LA, Chung VM et al (2015) Gene-mediated cytotoxic immunotherapy as adjuvant to surgery or chemoradiation for pancreatic adenocarcinoma. Cancer Immunol Immunother 64(6):727–736. https://doi.org/10.1007/s00262-015-1679-3
Wheeler LA, Manzanera AG, Bell SD et al (2016) Phase II multicenter study of gene-mediated cytotoxic immunotherapy as adjuvant to surgical resection for newly diagnosed malignant glioma. Neurooncology 18(8):1137–1145. https://doi.org/10.1093/neuonc/now002
Ji N, Weng D, Liu C et al (2016) Adenovirus-mediated delivery of herpes simplex virus thymidine kinase administration improves outcome of recurrent high-grade glioma. Oncotarget 7(4):4369–4378. https://doi.org/10.18632/oncotarget.6737
Ardiani A, Sanchez-Bonilla M, Black ME (2010) Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo. Cancer Gene Ther 17(2):86–96. https://doi.org/10.1038/cgt.2009.60
Willmon CL, Krabbenhoft E, Black ME (2006) A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing. Gene Ther 13(17):1309–1312. https://doi.org/10.1038/sj.gt.3302794
Akyürek LM, Nallamshetty S, Aoki K et al (2001) Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo. Mol Ther 3(5 Pt 1):779–786. https://doi.org/10.1006/mthe.2001.0315
Wei SJ, Chao Y, Hung YM et al (1998) S- and G2-phase cell cycle arrests and apoptosis induced by ganciclovir in murine melanoma cells transduced with herpes simplex virus thymidine kinase. Exp Cell Res 241(1):66–75. https://doi.org/10.1006/excr.1998.4005
Denny WA (2003) Prodrugs for gene-directed enzyme-prodrug therapy (suicide gene therapy). J Biomed Biotechnol 2003(1):48–70. https://doi.org/10.1155/S1110724303209098
Sato T, Neschadim A, Konrad M et al (2007) Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy. Mol Ther 15(5):962–970. https://doi.org/10.1038/mt.sj.6300122
Neschadim A, Wang JC, Lavie A et al (2012) Bystander killing of malignant cells via the delivery of engineered thymidine-active deoxycytidine kinase for suicide gene therapy of cancer. Cancer Gene Ther 19(5):320–327. https://doi.org/10.1038/cgt.2012.4
Neschadim A, Wang JC, Sato T et al (2012) Cell fate control gene therapy based on engineered variants of human deoxycytidine kinase. Mol Ther 20(5):1002–1013. https://doi.org/10.1038/mt.2011.298
Likar Y, Zurita J, Dobrenkov K et al (2010) A new pyrimidine-specific reporter gene: a mutated human deoxycytidine kinase suitable for PET during treatment with acycloguanosine-based cytotoxic drugs. J Nucl Med 51(9):1395–1403. https://doi.org/10.2967/jnumed.109.074344
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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Neschadim, A., Medin, J.A. (2019). Engineered Thymidine-Active Deoxycytidine Kinase for Bystander Killing of Malignant Cells. In: Düzgüneş, N. (eds) Suicide Gene Therapy. Methods in Molecular Biology, vol 1895. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8922-5_12
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DOI: https://doi.org/10.1007/978-1-4939-8922-5_12
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