1. Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes
- Author
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Junhui Wang, Daniel H. Fowler, Orlay Lopez-Perez, Ronan Foley, Jeffrey A. Medin, Bryan Au, Natalia Pacienza, Anton Neschadim, Sean Devine, C-J Lee, Matthew Scaife, and Elizabeth Scheid
- Subjects
CIENCIAS MÉDICAS Y DE LA SALUD ,TMPK ,Recombinant Fusion Proteins ,Genetic enhancement ,Antigens, CD19 ,Genetic Vectors ,GENE THERAPY ,Mice, SCID ,Protein Engineering ,Receptor, Nerve Growth Factor ,CD19 ,Biotecnología de la Salud ,Ciencias Biológicas ,Mice ,Transduction (genetics) ,Transformation, Genetic ,Mice, Inbred NOD ,Cell Line, Tumor ,Genetics ,medicine ,Animals ,Humans ,Leukemia-Lymphoma, Adult T-Cell ,Molecular Biology ,Severe combined immunodeficiency ,Cell Death ,biology ,Lentivirus ,HEK 293 cells ,Bioquímica y Biología Molecular ,medicine.disease ,Virology ,LENTIVIRUS ,Cell biology ,Raji cell ,HEK293 Cells ,Cell culture ,Ética relacionada con Biotecnología Médica ,AZT ,biology.protein ,Fabry Disease ,Molecular Medicine ,CELL-FATE CONTROL ,Nucleoside-Phosphate Kinase ,Zidovudine ,CIENCIAS NATURALES Y EXACTAS ,K562 cells - 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. Fil: Scaife, Matthew. University of Toronto; Canadá Fil: Pacienza, Natalia Alejandra. University Health Network. Ontario Cancer Institute; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Au, B. C. Y.. University Health Network. Ontario Cancer Institute; Canadá Fil: Wang, J. C. M.. University Health Network. Ontario Cancer Institute; Canadá Fil: Devine, S.. University of Toronto; Canadá Fil: Scheid, E.. Mc Master University; Canadá Fil: Lee, C. J.. University Health Network. Ontario Cancer Institute; Canadá Fil: Lopez Perez, O.. University Health Network. Ontario Cancer Institute; Canadá Fil: Neschadim, A.. University of Toronto; Canadá Fil: Fowler, D. H.. National Institutes of Health; Estados Unidos Fil: Foley, R.. Mc Master University; Canadá Fil: Medin, J. A.. University of Toronto; Canadá. University Health Network. Ontario Cancer Institute; Canadá
- Published
- 2012
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