技术资料

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors,genetic instability,and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells,transduced with these 2 ankyrin-1 promoter vectors,were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation,high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment),compared with negligible expression in myeloid and lymphoid lineages in blood,BM,spleen,and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus,modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer,stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases. View Publication

Patients with heart failure are predisposed to infections and anemia,possibly due to reduced hematopoiesis. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in heart failure,and it inhibits normal hematopoiesis,partly due to apoptosis through the effector molecule Fas. We examined bone marrow progenitor cells of mice with heart failure induced by acute myocardial infarction. The fraction of progenitor cells in mice with heart failure was only approximately 40% of control. Measured with in vitro clonal assays,the proliferative capacity of the progenitor cells in mice with heart failure was reduced to approximately 50% of control. Flow cytometry with specific markers revealed a threefold increase in apoptosis among progenitor cells from mice with heart failure. In these mice,TNF-alpha/Fas expression was increased in bone marrow natural killer (NK) and T cells,and these lymphocytes showed increased cytolytic activity in vitro against progenitor cells. We conclude that the TNF-alpha/Fas pathway in lymphocytes is activated in the bone marrow during heart failure,which may play a pathogenic role in the observed decrease in hematopoiesis. View Publication

Telomere length must be tightly regulated in highly proliferative tissues,such as the lymphohematopoietic system. Under steady-state conditions,the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc(-/-)) with critically short telomeres. Evaluation of self-renewal potential of mTerc(-/-) day-12 spleen colony-forming units demonstrated no alteration as compared with wildtype progenitors. However,the replating ability of mTerc(-/-) granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs,indicating a diminished capacity of late-generation mTerc(-/-) committed progenitors when forced to proliferate. Long-term bone marrow cultures of mTerc(-/-) bone marrow (BM) cells show a reduction in proliferative capacity; this defect can be mainly attributed to the hematopoietic,not to the stromal,mTerc(-/-) cells. In serial and competitive transplantations,mTerc(-/-) BM stem cells show reduced long-term repopulating capacity,concomitant with an increase in genetic instability compared with wildtype cells. Nevertheless,in competitive transplantations late-generation mTerc(-/-) precursors can occasionally overcome this proliferative impairment and reconstitute irradiated recipients. In summary,our results demonstrate that late-generation mTerc(-/-) BM cells with short telomeres,although exhibiting reduced proliferation ability and reduced long-term repopulating capacity,can still reconstitute myeloablated animals maintaining stem cell function. View Publication

CCAAT/enhancer binding proteins (C/EBPs) are a family of factors that regulate cell growth and differentiation. These factors,particularly C/EBPalpha and C/EBPepsilon,have important roles in normal myelopoiesis. In addition,loss of C/EBP activity appears to have a role in the pathogenesis of myeloid disorders including acute myeloid leukemia (AML). Acute promyelocytic leukemia (APL) is a subtype of AML in which a role for C/EBPs has been postulated. In almost all cases of APL,a promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) fusion protein is expressed as a result of a t(15;17)(q22;q12) chromosomal translocation. PML-RARalpha inhibits expression of C/EBPepsilon,whereas all-trans retinoic acid (tRA),a differentiating agent to which APL is particularly susceptible,induces C/EBPepsilon expression. PML-RARalpha may also inhibit C/EBPalpha activity. Thus,the effects of PML-RARalpha on C/EBPs may contribute to both the development of leukemia and the unique sensitivity of APL to tRA. We tested the hypothesis that increasing the activity of C/EBPs would revert the leukemic phenotype. C/EBPalpha and C/EBPepsilon were introduced into the FDC-P1 myeloid cell line and into leukemic cells from PML-RARA transgenic mice. C/EBP factors suppressed growth and induced partial differentiation in vitro. In vivo,enhanced expression of C/EBPs prolonged survival. By using a tamoxifen-responsive version of C/EBPepsilon,we observed that C/EBPepsilon could mimic the effect of tRA,driving neutrophilic differentiation in leukemic animals. Our results support the hypothesis that induction of C/EBP activity is a critical effect of tRA in APL. Furthermore,our findings suggest that targeted modulation of C/EBP activities could provide a new approach to therapy of AML. View Publication

Hematopoietic stem cells (HSC) are tightly regulated through,as yet,undefined mechanisms that balance self-renewal and differentiation. We have identified a role for the transcriptional coactivators CREB-binding protein (CBP) and p300 in such HSC fate decisions. A full dose of CBP,but not p300,is crucial for HSC self-renewal. Conversely,p300,but not CBP,is essential for proper hematopoietic differentiation. Furthermore,in chimeric mice,hematologic malignancies emerged from both CBP(-/-) and p300(-/-) cell populations. Thus,CBP and p300 play essential but distinct roles in maintaining normal hematopoiesis,and,in mice,both are required for preventing hematologic tumorigenesis. View Publication

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12,previously called stromal cell-derived factor 1,are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However,this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and,strikingly,is absent in circulating mature B cells,which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK),a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation,whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover,sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage-specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways. View Publication

A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report,we show that marrow cells from WAS protein (WASP)-deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells,including hematopoietic progenitors and stem cells (HSCs),have decreased homing capacities that were associated with a defect in adhesion to collagen. During development,HSCs migrate from the liver to the marrow and the spleen,prompting us to ask if a defect in HSC homing during development may explain the skewed X-chromosome inactivation in WAS carriers. Preliminary evidence has shown that,in contrast to marrow progenitor cells,fetal liver progenitor cells from heterozygous females had a random X-chromosome inactivation. When fetal liver cells from WASP-carrier females were injected into irradiated recipients,a nonrandom inactivation of the X-chromosome was found at the level of hematopoietic progenitors and HSCs responsible for the short- and long-term hematopoietic reconstitution. Therefore,the mechanism of the skewed X-chromosomal inactivation observed in WAS carriers may be related to a migration defect of WASP-deficient HSCs. View Publication

In a previous study of prevalidation,a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed,to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up,by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper,we describe an international blind trial designed to apply this model to the clinical neutropenia,by testing 20 drugs,including 14 antineoplastics (Cytosar-U,5-Fluorouracil,Myleran,Thioguanine,Fludarabine,Bleomycin,Methotrexate,Gemcitabine,Carmustine,Etoposide,Teniposide,Cytoxan,Taxol,Adriamycin); two antivirals (Retrovir,Zovirax,); three drugs for other therapeutic indications (Cyclosporin,Thorazine,Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin,Bleomycin,Etoposide,Fludarabine,5-Fluorouracil,Myleran,Taxol,Teniposide,Thioguanine,and Thorazine),IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine,Cyclosporin,Cytosar-U,Cytoxan,Gemcitabine,Indocin,Lindane,Methotrexate,Retrovir,and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD� View Publication

Embryonic stem cells can be induced in vitro,by coculture with the stromal line RP.0.10 and a mixture of interleukins 3,6,and 7,to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+,PgP-1+,c-kit+,Joro75-,B-220-,F4/80-,Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny. View Publication

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate all of the hematopoietic lineages in vivo and sustain the production of these cells for the life span of the individual. In the absence of reliable direct markers for HSCs,their identification and enumeration depends on functional long-term,multilineage,in vivo repopulation assays. The extremely low frequency of HSCs in any tissue and the absence of a specific HSC phenotype have made their purification and characterization a highly challenging goal. HSCs and primitive hematopoietic cells can be distinguished from mature blood cells by their lack of lineage-specific markers and presence of certain other cell-surface antigens,such as CD133 (for human cells) and c-kit and Sca-1 (for murine cells). Functional analyses of purified subpopulations of primitive hematopoietic cells have led to the development of several procedures for isolating cell populations that are highly enriched in cells with in vivo stem cell activity. Simplified methods for obtaining these cells at high yield have been important to the practical exploitation of such advances. This article reviews recent progress in identifying human and mouse HSCs and current techniques for their purification. View Publication

High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models. View Publication

View Publication