A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells

Background We have developed a novel system for expansion of gene-modified hematopoietic stem/progenitor cells to overcome the low efficiency of curre nt gene transfer methodology. This system involves 'selective amplifier gen es', that encode fusion proteins between the granulocyte colony-stimulating factor receptor (GCR) and the hormone-binding domain of estrogen receptor (ER). Hematopoietic progenitors expressing the chimeras showed estrogen-res ponsive growth in a controllable manner. However, endogenous estrogen may a ctivate the fusion proteins in vivo, depending on the hormonal status of th e subjects. Methods We replaced ER with a mutant receptor (TmR) which specifically bind s to 4-hydroxytamoxifen (Tm), to overcome limitations with wild-type ER. In terleukin-3 (IL-3)-dependent Ba/F3 cells and hematopoietic progenitor cells transduced with the resultant fusion proteins (GCRTmR and Delta GCRTmR) we re examined for ligand-inducible grouch. Results GCRTmR- and Delta GCRTmR-expressing Ba/F3 showed IL-3-independent g rowth in response to Tm, while the cells were unresponsive to estrogen at c oncentrations up to 10(-7)-10(-6) M. Furthermore, murine bone marrow cells transduced with GCRTmR and Delta GCRTmR formed colonies in methyl-cellulose medium in response to Tm, while virtually no colonies appeared with 10(-7) M estrogen or without cytokines. Conclusions These results suggest that influences of the endogenous estroge n can be almost eliminated by using the GCRTmR/Tm or Delta GCRTmR/ Tm syste m to expand gene-modified hematopoietic stem/progenitor cells. Copyright (C ) 1999 John Wiley & Sons, Ltd.