title: 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.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Scaife
authors.forename: N
authors.initials: N
authors.lastname: Pacienza
authors.forename: B C Y
authors.initials: BC
authors.lastname: Au
authors.forename: J C M
authors.initials: JC
authors.lastname: Wang
authors.forename: S
authors.initials: S
authors.lastname: Devine
authors.forename: E
authors.initials: E
authors.lastname: Scheid
authors.forename: C-J
authors.initials: CJ
authors.lastname: Lee
authors.forename: O
authors.initials: O
authors.lastname: Lopez-Perez
authors.forename: A
authors.initials: A
authors.lastname: Neschadim
authors.forename: D H
authors.initials: DH
authors.lastname: Fowler
authors.forename: R
authors.initials: R
authors.lastname: Foley
authors.forename: J A
authors.initials: JA
authors.lastname: Medin
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics*; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D000795:Fabry Disease / Q000235:genetics; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics*; D015202:Protein Engineering; D020800:Receptor, Nerve Growth Factor / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic; D015215:Zidovudine / Q000633:toxicity
chemical_list: D018941:Antigens, CD19; D020800:Receptor, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase