Gene Therapy (2002) 9, 452–462  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells N Eliopoulos 1 , A Al-Khaldi 2 , CM Beause ´jour 3 , RL Momparler 3 , LF Momparler 3 and J Galipeau 1,4 1 Lady Davis Institute for Medical Research, Department of Experimental Medicine, McGill University, Montreal, Canada; 2 Division of Cardiothoracic Surgery, McGill University Health Center, Montreal, Canada; 3 Department of Pharmacology, Universite ´ de Montre ´al, Centre de Recherche Pe ´diatrique, Ho ˆpital Ste-Justine, Montreal, Canada; and 4 Department of Medicine, Division of Hematology/Oncology, Jewish General Hospital, McGill University, Montreal, Canada 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 chemoresist- ance 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 com- prising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduc- tion 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 Keywords: cytidine deaminase; drug selectable marker; gene therapy; bone marrow stroma; cytosine arabinoside; retro- viral vector Introduction Transgene expression in a plurality of engineered target cells is an essential but challenging goal in many cell and gene therapy applications. One approach to achieve this would be to dominantly select and enrich ex vivo the cells concomitantly expressing a therapeutic transgene and a linked dominant selectable marker gene. Numerous investigators have employed dominant selection stra- tegies utilizing drug resistance genes, such as the prokar- yotic neomycin phosphotransferase II (Neo R ) gene, 1,2 the human multidrug resistance (MDR) gene, 3–5 the mutated human dihydrofolate reductase (DHFR) genes, 6,7 and the human O 6 -methylguanine-DNA-methyltransferase (MGMT) gene. 8,9 However, disadvantages have been noted with some drug selectable markers. For instance, cells modified with the prokaryotic Neo R gene may elicit an immune reaction in vivo, 10 MDR-transduced hemato- Correspondence: J Galipeau, Lady Davis Institute for Medical Research, 3755 Cote Ste Catherine Road, Montreal, Quebec, H3T 1E2 Canada Received 9 September 2001; accepted 8 January 2002 mixture of transduced and untransduced MSCs, the percent- age of retrovector-expressing cells could be increased to vir- tual purity (>99.5%) through in vitro drug selection with 1 M Ara-C. Increased selective pressure with 2.5 M 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 engine- ered MSCs. Moreover, engraftment and endothelial differen- tiation 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. Gene Therapy (2002) 9, 452–462. DOI: 10.1038/sj/gt/3301675 poietic stem cells, when transplanted in mice, can lead to a myeloproliferative syndrome, 5 and MGMT gene-modi- fied cells require selection with the DNA damaging alkylating agents. 11 Desirable features for a drug sel- ectable marker would include low or absent immuno- genicity, little or no collateral biochemical perturbation of engineered cells and lastly, chemical selection with drugs having low mutagenic activity. In previous studies, we cloned and expressed the human cytidine deaminase cDNA which consists of 910 bp encoding an 146 amino acid protein of 16.2 kDa. 12 Human cytidine deaminase (hCD) (EC 3.5.4.5) is an enzyme that catalyzes the deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. 13 It is normally expressed in a wide array of normal cells and tissues, especially liver and spleen. Increased expression of the endogenous hCD gene in malignant cells has been linked to chemotherapy resistant leukemias in humans. This observation led to the study of the hCD gene in chemoprotective gene therapy. Indeed, we have shown that the hCD drug resistance gene has promise for protecting normal hematopoietic cells from the dose- limiting myelotoxicity of anti-cancer drugs cytosine