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Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes

Abstract

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3′-azido-3′-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34+ cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies.

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Acknowledgements

We would like to especially thank the Fabry patients who donated bone marrow to facilitate these studies. This research was funded by a research operating grant from the Canadian Institutes of Health Research to Dr JA Medin. Funding for AN was provided by the CIHR Training Program in Regenerative Medicine (TPRM).

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Correspondence to J A Medin.

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Scaife, M., Pacienza, N., Au, B. et al. Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes. Gene Ther 20, 24–34 (2013). https://doi.org/10.1038/gt.2011.210

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