Bicistronic retroviral vectors for combining myeloprotection with cell-surface marking

• Published in: Gene therapy Vol. 6; no. 7; pp. 1222 - 1230
• Main Authors: HILDINGER, M, SCHILZ, A, ECKERT, H.-G, BOHN, W, FEHSE, B, ZANDER, A. R, OSTERTAG, W, BAUM, C
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing Group 01-07-1999
• Subjects: Actin; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; Biological and medical sciences; Biotechnology; CD34 antigen; Cell surface; Chemotherapy; Cytostatic agents; Drug Resistance, Multiple - genetics; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Gene expression; Gene therapy; Gene Transfer Techniques; Genetic Markers; Genetic Vectors - genetics; Health. Pharmaceutical industry; Hematopoietic stem cells; Humans; Industrial applications and implications. Economical aspects; internal ribosome entry sites; MDR1 protein; Multidrug resistance; P-Glycoprotein; Polymerase Chain Reaction; Progenitor cells; Receptor, Nerve Growth Factor - genetics; Retroviridae - genetics; Retrovirus; Stem Cells; Tumor Cells, Cultured; Antineoplastic agent; Toxicity; Retroviridae; Gene expression; Myeloid cell; Cell surface; Virus; Vertebrata; Chemotherapy; Mammalia; Secondary effect; Gene therapy; Protection; Vector
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3300942

We have developed a retroviral vector coexpressing the multidrug-resistance 1 (MDR1) cDNA for inducing cancer drug resistance and the truncated version of the low-affinity nerve growth factor receptor (DeltaLNGFR) for cell-surface marking of transduced cells. The vector is based on the FMEV backbone which mediates high levels of gene expression in hematopoietic cells. To achieve optimal expression levels of both cDNAs, untranslated regions from MDR1 and DeltaLNGFR were removed and three different connections were tested: retroviral splice signals, an internal ribosomal entry site (IRES) from encephalomyocarditis virus, and an internal promoter from the chicken beta-actin gene. As determined by two-color flow cytometry, the best correlation of the expression of both cDNAs was obtained using the vector SF1mSdelta which utilized retroviral splice signals for co-expression. Simultaneous expression of both cDNAs at the single cell level was also shown by confocal laser microscopy. Lymphoid and hematopoietic progenitor cells, including primary human CD34+ cells, transduced with SF1mSdelta acquired dominant multidrug resistance. Transduced primary CD34+ cells could be enriched in vitro based on expression of DeltaLNGFR, avoiding exposure to cytostatic agents. Thus, monitoring the selection of chemotherapy-resistant cells and analyzing their biological properties may be alleviated, both in vitro and in vivo.