Development of a Novel Selective Amplifier Gene for Controllable Expansion of Transduced Hematopoietic Cells

Abstract

AbstractTo overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)–dependent murine cell line, Ba/F3, the cells showed IL-3–independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Δ(5-195)GCRER, whose product lacks the extracellular G-CSF–binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Δ(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 untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, 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 signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.