In vivo expansion of gene-modified hematopoietic cells by a novel selective amplifier gene utilizing the erythropoietin receptor as a molecular switch.

Abstract

In vivo expansion of gene-modified cells would be a promising approach in the field of hematopoietic stem cell gene therapy. To this end, we previously developed a selective amplifier gene (SAG), a chimeric gene encoding the granulocyte colony-stimulating factor (G-CSF) receptor (GCR), as a growth-signal generator and the hormone-binding domain of the steroid receptor as a molecular switch. We have already reported that hematopoietic cells retrovirally transduced with the SAG can be expanded in a steroid-dependent manner in vitro and in vivo in mice and nonhuman primates. In this study, we have developed a new-generation SAG, in which the erythropoietin (EPO) receptor (EPOR) is utilized instead of the steroid receptor as a molecular switch. Two EPO-driven SAGs were constructed, EPORGCR and EPORMpl, containing the GCR and c-Mpl as a signal generator, respectively. First, to compare the steroid-driven and EPO-driven SAGs, Ba/F3 cells were transduced with these SAGs. Next, to examine whether GCR or c-Mpl is the more suitable signal generator of the EPO-driven SAG, human cord blood CD34(+) cells were transduced with the two EPO-driven SAGs (EPORMpl and EPORGCR). Finally, we examined the in vivo efficacy of EPORMpl in mice. Irradiated mice were transplanted with EPORMpl-transduced bone marrow cells followed by administration of EPO. The EPO-driven SAGs were shown to induce more rapid and potent proliferation of Ba/F3 cells than the steroid-driven SAGs. The EPORMpl induced more efficient EPO-dependent proliferation of the human cord blood CD34(+) cells than the EPORGCR in terms of total CD34(+) cell, c-Kit(+) cell, and clonogenic progenitor cell (CFU-C) numbers. In the transplanted mice the transduced peripheral blood cells significantly increased in response to EPO. The new-generation SAGs, especially EPORMpl, are able to efficiently confer an EPO-dependent growth advantage on transduced hematopoietic cells in vitro and in vivo in mice.