title: Cell surface tagging and a suicide mechanism in a single chimeric human protein.
abstract: Many therapeutic uses of gene-modified cells could benefit from inclusion of a surface marker for immunoselecting transduced cells. Another desired feature is a failsafe mechanism to ablate engineered cells if required. We describe here a system that combines a cell surface tag and an inducible apoptosis mechanism in a single protein. Spencer et al. (Curr. Biol. 1996;6:839-847) described an inducible cell suicide gene containing a myristoylation sequence, the human protein FKBP12, and the intracellular domain of Fas. Cells expressing this protein apoptose on treatment with a cell-permeable chemical dimerizing agent that binds two FKBP domains and cross-links the chimeric Fas proteins. We modified this system by anchoring a Fas-FKBP construct to the membrane with the extracellular and transmembrane domains of the low-affinity nerve growth factor receptor (LNGFR), thereby uniting cell surface tagging with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct apoptosed on exposure to a chemical dimerizer, AP1903 (Clackson et al., Proc. Natl. Acad. Sci. U.S.A. 1998;95:10437-10442). The LNGFR-tagged construct showed an unpredicted clear advantage over the myristoylation-anchored construct in its efficiency of signaling in HT1080 cells. This linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy. The use of gene-modified cells in basic research and clinical studies is enhanced by the use of a selectable surface marker for immunoselection of transduced cells. Another desired feature for gene and cell therapies is an inducible suicide system to eliminate transduced cells when necessary. Spencer et al. (Curr. Biol. 1996;6:839-847) described a potential failsafe mechanism whereby exposure of cells to a chemical dimerizing agent activates the Fas-mediated apoptotic pathway. In this system, the intracellular signaling domain of Fas is linked to one or more copies of the human protein FKBP12. Treatment of engineered cells with a cell-permeable chemical dimerizing agent that simultaneously binds to two FKBP domains cross-links the chimeric Fas protein and induces apoptosis. Here, we modify the system by anchoring a Fas-FKBP construct to the membrane with the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR), to unite cell surface tagging of transduced cells with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct undergo apoptosis on exposure to a chemical dimerizer, AP1903. A linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy.
authors.affiliation: ARIAD Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Amara
authors.forename: N L
authors.initials: NL
authors.lastname: Courage
authors.forename: M
authors.initials: M
authors.lastname: Gilman
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002460:Cell Line / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D020104:Immunophilins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020800:Receptor, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D022021:Tacrolimus Binding Proteins; D014161:Transduction, Genetic; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: D020800:Receptor, Nerve Growth Factor; D011994:Recombinant Proteins; D019014:fas Receptor; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins