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Exposure to hypomethylating agent, 5-azacytidine, may improve iCasp9 suicide gene therapy for treating GvHD in allografts

Gene Therapy volume 23, pages 664–672 (2016)Cite this article

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Abstract

Anti-tumor cellular immunotherapies that implement a suicide gene system can limit potential undesirable effects. In a haplo-identical bone marrow transplant clinical trial, over 90% of iCaspase-9-expressing cells were eradicated after AP1903 exposure, and signs of graft-versus-host disease disappeared. Nevertheless, low numbers of genetically modified T cells survived this treatment. We studied genetically modified cell lines (GMCL) that carried a dual iCaspase-9/ΔCD19 DNA construct (ΔCD19=truncated CD19). With AP1903 exposure, a low percentage of cells (1.47±0.67%; n=5 replications) persisted in vitro. Repeated exposures to increasing AP1903 doses generated low (GMCLLR) and high AP1903-responders (GMCLHR), which expressed different levels of surface ΔCD19 and intracellular iCaspase-9. Compared with GMCLHR, GMCLLR exhibited higher methylation of 5′-long-terminal repeat (LTR) promoters, both in the number of sequences with at least one methylated CpG (16 vs 51.5%, respectively) and in the number of CpG islands (1.2 vs 8.9%, respectively). Four days of 5-azacytidine exposure reduced methylation and increased ΔCD19 and iCaspase-9 expression. Interestingly, LTR demethylation restored GMCLLR sensitivity to AP1903 by 24.3-fold (1.8 vs 43.8%) without affecting GMCLHR. We showed that 5′-LTR-methylation inhibited transgene expression and caused AP1903 hypo-responsiveness. Treating with a hypomethylating agent restored AP1903 sensitivity. This approach can be applied in further clinical trials to improve iCaspase-9 response if low response is detected.

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Acknowledgements

We thank Pr Malcolm Brenner (Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, USA) for providing research grade of packaging cell line and retroviral supernatant. We also thank Pr David Spencer (Bellicum Pharmaceuticals, Houston, USA) for providing research grade AP1903 Chemical Inducer Dimerizer and for revising this manuscript. This study was supported by the ‘Programme Hospitalier de Recherche Clinique’ (PHRC #950898), the ‘Ligue Nationale contre le Cancer’ (2013), Etablissement Français du sang (APR 2013) and Région de Franche-Comté (Grant 2013); it was co-funded, in part, by the 2013/A02 tender of the Association Capucine and the Société Française de Greffe de Moelle—Thérapie Cellulaire (SFGM-TC).

Author contributions

EB-R executed all experiments, including cell cultures, flow cytometry and western blotting, and she wrote the original draft of the manuscript. CG, II and J-MC assisted with retroviral transductions, GMTC production and AP1903 resistance. A-MM, PT and CB contributed to improving the manuscript and provided final approval. ED and FL provided relevant information of clinical interest and information on future clinical trials. CF and MD initiated and designed the study, participated in every step of the study, managed the whole project and wrote the manuscript.

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Authors and Affiliations

  1. Laboratoire de Thérapeutique Immuno-Moléculaire et cellulaire des cancers, INSERM UMR1098, Etablissement Français du Sang Bourgogne Franche-Comté, Université de Franche-Comté, SFR FED4234, Besançon, France

    E Bôle-Richard, C Gamonet, J-M Certoux, I Idirene, F Larosa, E Deconinck, P Tiberghien, C Borg, C Ferrand & M Deschamps

  2. Hematology Department, CHU Jean Minjoz, Besançon, France

    F Larosa & E Deconinck

  3. Bellicum pharmaceuticals, Houston, TX, USA

    A-M Mosseley

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  1. E Bôle-Richard
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Correspondence to M Deschamps.

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Bôle-Richard, E., Gamonet, C., Certoux, JM. et al. Exposure to hypomethylating agent, 5-azacytidine, may improve iCasp9 suicide gene therapy for treating GvHD in allografts. Gene Ther 23, 664–672 (2016). https://doi.org/10.1038/gt.2016.39

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