DEVELOPMENT OF A NOVEL SELECTIVE AMPLIFIER GENE FOR CONTROLLABLE EXPANSION OF TRANSDUCED HEMATOPOIETIC-CELLS

To overcome the low efficiency of gene transfer into hematopoietic cel ls, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding th e fusion protein between the granulocyte colony-stimulating factor rec eptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen re ceptor (ER) as a selective amplifier gene. Use of the intracellular si gnaling pathway of G-CSFR was considered to be appropriate, because G- CSF has the ability not only to stimulate the neutrophil production, b ut also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estro gen/ER-HBD system was used as a molecular switch in this study. When t he GCRER gene was expressed in the interleukin-3 (IL-3)-dependent muri ne cell line, Ba/F3, the cells showed IL-3-independent growth in respo nse to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with t he Delta(5-195)GCRER, whose product lacks the extracellular G-CSF-bind ing domain, did not respond to G-CSF, but retained the ability for est rogen-dependent growth. Further, murine bone marrow cells transduced w ith the GCRER or Delta(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransd uced murine bone marrow cells. It is noteworthy that erythroid colonie s were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythro poietin, suggesting that the signals from the G-CSFR portion of the ch imeric molecules do not preferentially induce neutrophilic differentia tion, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth s ignal predominates and that the population of transduced stem cells ex pands upon estrogen treatment, even if some of the cells enter the dif ferentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoi etic stem cells. (C) 1997 by The American Society of Hematology.