久米 晃啓

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

We previously developed "selective amplifier genes (SAGs)" which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34(+) cells than the GCR-type SAG. One mutant Mpl-type SAG (Delta GCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.

BACKGROUND: Composite tissue allografts are unique because they provide the vascularized bone marrow with stroma, which is the supportive microenvironment. In this study, we investigated the beneficial effect of donor-derived bone marrow cells within the long-surviving recipient rats after limb transplantation. METHODS: Green fluorescent protein (GFP) transgenic rats developed for paramount cell marking were donors, and wild Wistar rats were recipients. Orthotopic hind-limb transplantation was performed using a microsurgical technique. Tacrolimus (1.0 mg/kg) was intramuscularly injected for 14 days postoperatively. The skin graft from GFP donor onto the GFP recipient was performed as a control. Flow cytometric analyses of recipient peripheral blood and bone marrow were carried out at 4 to 6 days, 18 to 21 days, 6 weeks, and 2, 4, 6, 9, and 12 months after transplantation. RESULTS: The rats that received tacrolimus therapy achieved prolonged composite graft acceptance more than 12 months, whereas GFP skin grafts were rejected at 47 days under the same immunosuppressive protocol. Numerous GFP lymphocytes and granulocytes were detected within the recipient bone marrow for the first 6 weeks post limb transplantation. These cells remained relatively stable for more than 12 months. CONCLUSIONS: The results showed that donor-derived hematopoietic stem cells engrafted in recipient bone marrow and differentiated to lymphocytes and granulocytes after limb transplantation. The vascularized bone marrow, transplanted as a part of the hind limb, could have contributed to mixed chimerism and worked as the bone-marrow source in the recipients.

Background The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning. Methods Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34(+) cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy x 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells. Results Although 13-37% of transduced cells contained the GFP provirus and 11-13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5-10%) of clonogenic progenitors contained the provirus and fluoresced exvivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes. Conclusions Low levels of GFP-transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP this block an treatment ex vivo and in vivo. Copyright (C) 2002 John Wiley Sons, Ltd.