I Could Die for You: New Prospects for Suicide in Gene Therapy (2007)

Molecular therapy in the year 2050: the editor invites authors to submit papers that describe the transfer of complex genetic information containing complete sets of developmental instructions for the control of cell fate in vivo. By this time the technological basis for such multigene transfer will have been established, with the creation of new generations of versatile, receptor-targeted, episomal gene vectors characterized by a large genome capacity and persistent gene expression. Safe and efficient delivery of genetic information into cells no longer represents a limitation. Systems biology will have allowed more precise predictions of cell behavior in multicellular organisms and of gene interactions in response to environmental stimuli. A remaining challenge at this (unfortunately still distant) stage of the development of the field is the design of “intelligent episomes” that comprise entire gene networks that allow multimodular manipulation of cell behavior. Among the modules used for this purpose are several that fulfill only one aim: to cause the engineered cell to self-destruct in case of misbehavior. A gene encoding the melancholic message “I could die for you,” or, in less poetic terms: targeted cell death or conditional suicide. This is not pure science fiction. Certainly some of the selectable marker genes used in the year 2050 will rely on discoveries made today. Although suicide gene therapy has made its way into clinical trials beyond phase I/II1Tiberghien P Use of suicide genes in gene therapy.J Leukoc Biol. 1994; 56: 203-209Crossref PubMed Scopus (75) Google Scholar,2Bonini C Ferrari G Verzeletti S Servida P Zappone E Ruggieri L et al.HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.Science. 1997; 276: 1719-1724Crossref PubMed Scopus (1035) Google Scholar,3Recchia A Bonini C Magnani Z Urbinati F Sartori D Muraro S et al.Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells.Proc Natl Acad Sci U S A. 2006; 103: 1457-1462Crossref PubMed Scopus (167) Google Scholar,4Portsmouth D Hlavaty J Renner M Suicide genes for cancer therapy.Mol Aspects Med. 2007; 28: 4-41Crossref PubMed Scopus (140) Google Scholar and has also been considered as an emergency brake in the case of tumor induction by insertional mutagenesis,5Frank O Rudolph C Heberlein C von Neuhoff N Schrock E Schambach A et al.Tumor cells escape suicide gene therapy by genetic and epigenetic instability.Blood. 2004; 104: 3543-3549Crossref PubMed Scopus (57) Google Scholar,6Blumenthal M Skelton D Pepper KA Jahn T Methangkool E Kohn DB Effective suicide gene therapy for leukemia in a model of insertional oncogenesis in mice.Mol Ther. 2007; 15: 183-192Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar the perfect suicide gene has yet to be identified. The therapeutic value of such a gene is based on many parameters: it should not alter cell growth in the absence of a suicide-inducing drug, it should not induce the expression of immunogenic epitopes, and the drug used to trigger cell death should not be toxic in other cells. A possible breakthrough toward this goal is presented in the article by Medin's group published in this issue of Molecular Therapy,7Sato T Neschadim A Konrad M Fowler DH Lavie A Medin JA Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy.Mol Ther. 2007; 15: 962-970Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar and it is quite possible that the work described in this article will continue to be referenced in manuscripts submitted in the year 2050 that describe work using complex episomes for the control of cell behavior. So what did Medin and colleagues do? They considered that the prodrug 3′-azido-3′-deoxythymidine, better known as AZT and widely used in HIV therapy, must be converted to the toxic triphosphate AZT-TP by cellular enzymes for it to wreak its havoc on the cell. The rate-limiting step in this process is catalyzed by thymidylate kinase (TmpK). To enhance the potency of the drug, Sato et al.7Sato T Neschadim A Konrad M Fowler DH Lavie A Medin JA Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy.Mol Ther. 2007; 15: 962-970Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar designed point mutants into the TmpK gene so as to accelerate AZT phosphorylation, thus efficiently converting this fairly safe prodrug into a strong antimetabolite that effectively kills cells into which it is introduced. Subsequent mechanistic studies indicated that cellular apoptosis was triggered in response to expression of the mutant TmpK and exposure to AZT by activation of caspase-3 and disruption of the mitochondrial inner membrane potential. An interesting aside to this finding, as suggested by the authors, is that it may provide an explanation for the toxic mitochondrial myopathy observed in some patients subsequent to long-term treatment with AZT. Another important observation of this study is the finding that this mechanism of cell death did not necessarily require cell division. This may be of particular importance when attempting to destroy quiescent tumor stem cells or other transplanted cells that may cause harm in the body even in the absence of a high proliferative index. Underlining the potency of the mutant TmpK/AZT suicide team, proof-of-concept experiments revealed efficient cell death in transduced immortal and primary T cells studied in vitro and a transduced K562 cell tumor model evaluated in immunodeficient mice. This suggests that this particular suicide gene/prodrug combination could be useful to eliminate either transplanted T cells in case of an attack against healthy tissue (so-called graft-versus-host disease) or tumor cells (that may arise as a complication of cell therapy or that may be directly targeted in vivo). Of course, a few questions remain unanswered, but these are probably the subject of ongoing investigations. These include the long-term effects of ectopic expression of mutant TmpK on cell growth and differentiation, the degree of bystander killing of neighboring cells that do not contain the transgene, the ability to counteract acute and chronic graft-versus-host disease subsequent to genetic modification of T cells, the ability to enrich cells coexpressing mutant TmpK and a positive selection marker using clinically applicable selection systems, and the frequency of genetic and epigenetic revertants or other forms of intrinsic resistance (which have been described using other suicide genes).5Frank O Rudolph C Heberlein C von Neuhoff N Schrock E Schambach A et al.Tumor cells escape suicide gene therapy by genetic and epigenetic instability.Blood. 2004; 104: 3543-3549Crossref PubMed Scopus (57) Google Scholar,8Thomis DC Marktel S Bonini C Traversari C Gilman M Bordignon C Clackson T A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease.Blood. 2001; 97: 1249-1257Crossref PubMed Scopus (168) Google Scholar,9Chalmers D Ferrand C Apperley JF Melo JV Ebeling S Newton I et al.Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene.Mol Ther. 2001; 4: 146-148Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Indeed, the fact that a single point mutation is sufficient to create a suicidal (and potentially nonimmunogenic) version of TmpK implies that another counteracting single point mutation might lead to loss of its suicide activity. A malicious “I will not die for you after all” may be the unfortunate message encoded by such an escape mutant. As suggested in the present and earlier reports,5Frank O Rudolph C Heberlein C von Neuhoff N Schrock E Schambach A et al.Tumor cells escape suicide gene therapy by genetic and epigenetic instability.Blood. 2004; 104: 3543-3549Crossref PubMed Scopus (57) Google Scholar,10Uckert W Kammertons T Haack K Qin Z Gebert J Schendel DJ et al.Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.Hum Gene Ther. 1998; 9: 855-865Crossref PubMed Scopus (92) Google Scholar this potential drawback could be addressed by the coexpression of a second suicide gene allowing targeted cell elimination. Nevertheless, the study by Sato et al.introduces a promising new approach for targeted induction of cell death with proof-of-concept analyses in model systems. The mutant TmpK/AZT suicide combination may thus prove to be a useful new tool for future gene and cell therapy.