title: Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.
abstract: Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.
authors.affiliation: Lady Davis Institute for Medical Research, Department of Experimental Medicine, McGill University, Montreal, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Eliopoulos
authors.forename: A
authors.initials: A
authors.lastname: Al-Khaldi
authors.forename: C M
authors.initials: CM
authors.lastname: Beauséjour
authors.forename: R L
authors.initials: RL
authors.lastname: Momparler
authors.forename: L F
authors.initials: LF
authors.lastname: Momparler
authors.forename: J
authors.initials: J
authors.lastname: Galipeau
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D015415:Biomarkers; D001854:Bone Marrow Cells; D002469:Cell Separation; D003561:Cytarabine / Q000494:pharmacology; D003564:Cytidine Deaminase / Q000235:genetics*; D004351:Drug Resistance; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009369:Neoplasms / Q000628:therapy*; D012190:Retroviridae / Q000235:genetics; D017154:Stromal Cells; D014161:Transduction, Genetic / Q000379:methods; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D015415:Biomarkers; D008164:Luminescent Proteins; D003561:Cytarabine; D049452:Green Fluorescent Proteins; D003564:Cytidine Deaminase