Hitoshi Hibino

AML1(-/-) embryos do not express certain hematopoiesis-related gene transcripts including those of the PU.1 gene.

The AML1 and PEBP2β/CBFβ genes encode the DNA-binding and non-binding subunits, respectively, of the heterodimeric transcription factor, PEBP2/CBF. Targeting each gene results in an almost identical phenotype, namely the complete lack of definitive hematopoiesis in the fetal liver on embryonic day 11.5 (E11.5). We examined and compared the expression levels of various hematopoiesis-related genes in wild type embryos and in embryos mutated for AML1 or PEBP2β/CBFβ. The RNAs were prepared from the yolk sacs of E9.5 embryos, from the aorta-gonad- mesonephros regions of E11.5 embryos and from the livers of E11.5 embryos and RT–PCR was performed to detect various gene transcripts. Transcripts were detected for most of the hematopoiesis-related genes that encode transcription factors, cytokines and cytokine receptors, even in tissues from homozygously targeted embryos. On the other hand, PU.1 transcripts were never detected in any tissue of AML1(−/−) or PEBP2β/CBFβ(−/−) embryos. In addition, transcripts for the Vav, flk-2/flt-3, M-CSF receptor, G-CSF receptor and c-Myb genes were not detected in certain tissues of the (−/−) embryos. The results suggest that the expression of a particular set of hematopoiesis-related genes is closely correlated with the PEBP2/CBF function.

GM-CSF and B7-1 (CD80) co-stimulatory signals co-operate in the induction of effective anti-tumor immunity in syngeneic mice

B7‐1 (CD80) co‐stimulatory molecule gene–transduced Lewis lung carcinoma (LLC) cells (LLC/B7 cells) resulted in remarkable loss of tumorigenicity in syngeneic C57BL/6 mice (87.5% rejection) compared to B7‐negative, wild‐type LLC (LLC/wt) cells (0% rejection). However, mice that had rejected LLC/B7 cells developed almost no systemic immunity protective against challenge with wild‐type tumor cells after 4 weeks (11.8% rejection). Enhancement of MHC class I (H‐2Kb) expression of LLC/B7 cells with in vitro interferon‐γ treatment did not result in enhancement of protective immunity. In vivo depletion assay revealed that abrogation of tumorigenicity in LLC/B7 depended on CD8+ T cells but not on CD4+ T cells. However, vaccination of C57BL/6 mice with irradiated LLC cells transduced with GM‐CSF (LLC/GM) led to the induction of potent, specific immunity against challenge with the LLC/wt cells after 2 weeks (80.8% rejection). Next, we established a double transfectant of LLC cells expressing both B7‐1 and GM‐CSF (LLC/GM + B7). The tumorigenicity of these clonal cells was also remarkably suppressed (90% rejection) to the same degree as LLC/B7, whereas that of LLC/GM was not suppressed (0% rejection). Interestingly, mice that had rejected LLC/GM+B7 cells developed enhanced protective immunity against challenge with LLC/wt cells after 4 weeks (55.6% rejection) compared to the results of LLC/B7 cells (11.8%). To evaluate whether co‐expression of GM‐CSF and B7‐1 enabled the tumor cells to activate cytotoxic T cells more efficiently than B7‐1 alone, we performed an in vitro killing assay. We found that immunization with LLC/GM+B7 cells resulted in a 3‐fold stronger cytotoxic response than that with LLC/B7. Our data indicate that co‐transfection of the B7‐1 co‐stimulatory molecule and GM‐CSF genes may be more effective for the induction of stronger protective immunity in this experimental system. Int. J. Cancer 73:556–561, 1997.

MHC (major histocompatibility complex)-DRB genes and polymorphisms in common marmoset.

Abstract. A New World monkey, the common marmoset (Callithrix jacchus), will be used as a preclinical animal model to study the feasibility of cell and gene therapy targeting immunological and hematological disorders. For elucidating the immunogenetic background of common marmoset to further studies, in the present study, polymorphisms of MHC-DRB genes in this species were examined. Twenty-one Caja-DRB exon 2 alleles, including seven new ones, were detected by means of subcloning and the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods followed by nucleotide sequencing. Based on the alignment of these allele sequences, we designed two pairs of specific primers and established a PCR-SSCP method for DNA-based histocompatibility typing of the common marmoset. According to the family segregation data and phylogenetic analyses, we presumed that Caja-DRB alleles could be classified into five different loci. Southern blotting analysis also supported the existence of multiple DRB loci. The patterns of nucleotide substitutions suggests that positive selection operates in the antigen-recognition sites of Caja-DRB genes.

Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectors

Abstract: To establish a new non‐human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti‐human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid‐specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c‐kit mAbs could be used to purify the marmoset haematopoietic progenitor cells.

Significant expression of G‐CSF‐induced gene‐1 (GIG‐1) protein in myeloid cells and NK cells

The granulocyte colony‐stimulating factor (G‐CSF)‐induced gene, GIG‐1, was originally cloned from G‐CSF‐stimulated bone marrow mononuclear cells obtained from a patient with chronic myelogenous leukemia (CML). We have characterized the GIG‐1 gene and its protein product. Expression of GIG‐1 mRNA was elevated by treatment with G‐CSF in normal bone marrow mononuclear cells, as well as in some cases of blast cells obtained from patients with acute myelogenous leukemia (AML) and CML. Western blot analysis with antiGIG‐1 peptide antiserum showed the molecular massofGIG‐1 product was about 17 kDa. Immuno‐staining of the hematopoietic cells demonstrated that GIG‐1 product was mainly localized to the cytoplasm of both myeloid and natural killer (NK) cells. These results suggested that GIG‐1 protein is an integral component that is accumulated during the differentiation of myeloid cells toward the stage of mature neutrophils. Expression of GIG‐1 gene in mature neutrophils was tightly regulated and reactivation of GIG‐1 gene by G‐CSF in mature neutrophils may represent a compensation process for the protein lost through the activation of these cells, thus implying an important role for this protein in host defense mechanisms. J. Leukoc. Biol. 65: 109–116; 1999.

Hematological Aspects of Common Marmoset Monkey Transplanted with Autologous MDR1 Gene Transduced Peripheral Blood Stem Cells

We have developed a small primate model system for human gene therapy using the common marmoset to evaluate the safety and efficacy of gene therapeutic approaches. We demonstrated that retroviral transduction of multidrug resistance gene (MDR1) into G-CSF-mobilized peripheral blood progenitor cells (PBPC) was improved by culturing PBPC with the mixed populations of bone marrow stroma cells and viral producer cells. We performed autologous PBPC transplantation using MDR1- transduced PBPC in two marmosets. The transduction efficiency to PBPC was 5.9–6.7% and provirus DNA could be detected in peripheral blood granulocytes and lymphocytes by PCR method in all gene transduced marmoset maximally up to 412 days after transplantation. MDR1-transduced autologous PBPC were tranasplanted to irradiated common marmosets and reconstituted their own hematopoiesis. Provirus MDR1 DNA could be detected in peripheral blood granulocytes and lymphocytes by PCR method in all gene transduced marmosets. Replication competent retrovirus was not detected in all marmosets by S+L− assay. Our preclinical stem/progenitor gene therapy system using marmoset is considered to be helpful for the development of human gene therapy.

ANALYSIS OF BONE MARROW STROMAL CELL TRANSFERRED BACTERIAL β - GALACTOSIDASE GENE BY PIXE

PIXE, Particle Induced X-ray Emission, is a Powerful, multi-elemental analysis method which has many distinguishing features and has been used in varies research fields. Recently the method of applying baby cyclotrons for nuclear medicine to PIXE has been developed. This enables us to study biomedical phenomena from the physical point of view. Mouse bone marrow stromal cells were transferred bacterial β -galactosidase gene (LacZ gene) by murine retroviral vectors. Analysis of the bone marrow stromal cells with the LacZ gene by PIXE revealed remarkable changes of intracellular trace elements compared with the normal control cells. These results indicate that gene transfer by retroviral vectors may bring about a dynamic change of intracellular circumstances of the target cell.

Common Marmoset as a New Preclinical Animal Model for Human Gene Therapy of Hematological Disorders

For the purpose of secured and effective introduction of gene therapeutic approaches to human hematological disorders, we have been focusing on the common marmoset, a small non-human primates as a target preclinical animal for gene transfer. Here we characterize the common marmoset bone marrow (MBM) progenitor cells in vitro and investigate whether these cells respond to the human cytokines and are transduced by retrovirus vectors. Namely, we screened human cytokines (erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, IL-6, IL-11, stem cell factor (SCF), and thrombopoietin(TPO)) to examine their stimulating activities on MBM progenitor cells by colonogenic assay in methyl-cellulose. These human cytokines were demonstrated to have significant effects on MBM progenitor cells and stimulated the colony forming activity of each lineage dose-dependently. We then studied LacZ gene transfer efficiencies into MBM progenitor cells by retrovirus vector in the presence of human IL-3, IL-6 and SCE By using the mixed cell populations of MBM stromal cells and retrovirus producer cells, the transduction efficiency to CFU-GM (CFU-GEMM) increased significantly. Our results suggest that the marmoset would be useful as a preclinical animal to evaluate the effectiveness and safety of new gene therapy vector sytems for hematological disorders.