UMLS:C0023418 - FACTA Search

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Query: UMLS:C0023418 (leukemia)
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Gaucher disease is a leading candidate for somatic gene therapy using bone marrow (BM) cells as target tissue. Towards this end, we have constructed a retroviral vector (LG) in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). Day 12 to 14 colony-forming unit-spleen progenitor cells were infected by the LG virus with a 100% efficiency, and GC messenger RNA (mRNA) and protein were detected in the progeny of these cells. Tissues from long-term reconstituted mice analyzed 8 months posttransplantation with LG-infected BM contained the intact provirus at greater than 1 copy per cell, indicating effective infection of hematopoietic stem cells. Human GC mRNA generated by the viral LTR was detected in macrophages as well as other hematopoietic cells. Enzyme activity was increased fivefold and twofold in macrophages from BM and spleen, respectively, and could be precipitated with an antibody specific for human GC. Immunohistochemical analysis detected the human GC protein in 81% of the macrophages from five recipient mice. These data indicate that, after transduction of hematopoietic stem cells, the LG vector is capable of directing expression of human GC in the majority of macrophages from long-term reconstituted mice and producing enzyme levels comparable with endogenous mouse activity, suggesting that this virus may be useful in the treatment of Gaucher disease.
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PMID:High levels of human glucocerebrosidase activity in macrophages of long-term reconstituted mice after retroviral infection of hematopoietic stem cells. 162 94

We are studying the transfer and expression by retroviral vectors of the human glucocerebrosidase (GC) gene into bone marrow cells as a model of gene therapy for genetic diseases of hematopoietic cells. A simple retroviral vector (G2) was developed that contains a normal human GC cDNA under the control of the Moloney murine leukemia virus long-terminal repeat (LTR) enhancer/promoter. Murine bone marrow was transduced with the G2 vector and maintained in long-term bone marrow culture (LTBMC). Expression of the human GC gene in the transduced murine LTBMC cells exceeded the level of endogenous murine GC mRNA. Murine bone marrow cells were also transduced with G2 and transplanted into irradiated syngeneic recipients. High levels of GC gene transfer and expression were seen in day-12 CFU-S foci, and to a lesser extent in the hematopoietic organs 4 months after gene transfer/bone marrow transplant (BMT). Human bone marrow, from a patient with Gaucher disease, was also used in studies of GC gene transduction. Gene transfer into 35-40% of the Gaucher hematopoietic progenitor cells was achieved, following prestimulation of the marrow with recombinant hematopoietic growth factors. Equal rates of gene transfer were obtained using either total marrow mononuclear cells or progenitor cells enriched 100-fold by immunomagnetic bead separation. GC gene transduction corrected the enzymatic deficiency of the Gaucher marrow. Our results demonstrate the potential utility of retroviral vector-mediated gene transfer for gene therapy of Gaucher disease. Current efforts are aimed at achieving more consistent in vivo GC expression in the murine BMT model and demonstrating transduction of pluripotent human hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Toward gene therapy for Gaucher disease. 191 28

A retroviral vector (GTN) in which the glucocerebrosidase (GCase) cDNA is driven by the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) was tested for transfer efficiency and expression of the GCase gene in long-term reconstituted mice. Eleven W/Wv mice were transplanted with unselected GTN-infected bone marrow cells and 10 of these mice were analyzed 3 months later. Seven of these 10 mice (70%) contained the intact proviral genome in bone marrow, spleen, and thymus. Of these 7,3 mice contained a high-copy number of the provirus in all the hematopoietic tissues tested. The mice contained anywhere from one to four proviral integration sites that were the same in all three tissues, indicating that these mice have been repopulated by one or more transduced multipotential hematopoietic stem cells. Five months after transplantation, bone marrow from the eleventh mouse was transplanted into secondary recipient animals. The secondary recipients contained the intact proviral genome in the bone marrow, spleen, thymus, and macrophages 4 months after the secondary transplantation. This further supports the conclusion that hematopoietic stem cells have indeed been targeted. Human GCase RNA was detected in all 7 mice containing the proviral DNA. These results demonstrate expression of the human GCase gene in the progeny of repopulating hematopoietic stem cells of mice following gene transfer.
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PMID:Expression of human glucocerebrosidase in long-term reconstituted mice following retroviral-mediated gene transfer into hematopoietic stem cells. 208 Nov 94

A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.
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PMID:Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer. 229 79

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, we developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py+/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hematopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeat (Mo-MLV LTR) and levels of Py+/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection (by means of the polymerase chain reaction) of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py+/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity had been normalized.
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PMID:Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease. 231 24

Gaucher disease (GD), one of the most common inherited metabolic disorders, is an excellent candidate for gene therapy using hematopoietic stem cells as targets. Animal models have demonstrated the feasibility of introducing the human glucocerebrosidase (GC) gene into hematopoietic progenitors with long term expression using a variety of retroviral vectors. We have previously demonstrated the expression and integration of the human GC gene in mouse hematopoietic progenitors and their progeny 4-8 months post transplant in primary recipients using the retroviral vector MFG-GC. We now demonstrate enzyme expression in peripheral blood lymphocytes of secondary recipients more than 12 months post transplantation. We also show a transduction efficiency of up to 95% in colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from transduced CD34+ cells from a variety of sources, using a centrifugation promoted infection protocol. Transduction has also been documented in long term culture initiating cells (LTCIC) from the same transduced CD34+ cells. These data indicate efficient transduction of mouse hematopoietic progenitors as well as human CD34+ cells using the retroviral vector MFG-GC.
Leukemia 1995 Oct
PMID:Long-term expression of the glucocerebrosidase gene in mouse and human hematopoietic progenitors. 747 11

We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.
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PMID:Myelosuppressive conditioning improves autologous engraftment of genetically marked hematopoietic repopulating cells in dogs. 753 34

Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.
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PMID:Retroviral-mediated gene expression in human myelomonocytic cells: a comparison of hematopoietic cell promoters to viral promoters. 757 92

The retroviral vector LGSN, in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was tested for expression in the murine myelomonocytic leukemia cell line M1 before and after induction of differentiation with interleukin-6 (IL-6). Southern analysis of the seven transduced clones selected for neomycin resistance in Geneticin (G-418 sulfate) demonstrated one to eight copies of intact provirus with rearrangements in only two clones. Absolute levels of human GC RNA and protein increased with increased copy numbers of provirus in the clones. Upon induction with IL-6 of the seven transduced clones to the macrophage phenotype, there was no significant change, overall, in RNA levels but some increase in human GC protein levels could be detected. Although this was the average trend, considerable clonal variation in RNA and protein levels was observed upon induction. Transduction of the M1 cells did not interfere with the ability of the cells to differentiate from blasts to macrophages as seen by the appearance of membrane receptors for the constant region of immunoglobulins (Fc gamma RI) and lysozyme production in the differentiated M1 cells. Thus, the M1 cell line can be used for testing retroviral vector expression in myeloid lineages at early and late stages of differentiation. This rapid in vitro testing of potential retroviral vectors will be beneficial for gene therapy of disorders that affect differentiated macrophages such as Gaucher's disease.
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PMID:Evaluation of expression of transferred genes in differentiating myeloid cells: expression of human glucocerebrosidase in murine macrophages. 768 78

To limit the genetic heterogeneity of schizophrenia, this study focused on the widely extended pedigrees of Ashkenazi Jewish schizophrenia probands. The hypothesis posed is that the increased prevalence among the Ashkenazim of the rare lysosomal enzyme disorders, Tay Sachs disease (TDS), caused by low levels of hexosaminidase A, and Gaucher's disease (GD), caused by low levels of glucocerebrosidase, might contribute to the demonstrated increased vulnerability to schizophrenia in this ethnic group. Signs and symptoms characterizing the candidate illnesses were systematically queried by the family history method. Rates and relative risks for symptoms characterizing these disorders and for several nonautosomal illnesses associated with TSD and/or GD (i.e., amyotrophic lateral sclerosis and Hodgkin's disease, leukemia and lymphoma) are significantly elevated in the schizophrenia pedigrees, compared to controls. The conditions with elevated rates and risks have been associated with chromosomal regions 1q21 and 15q23-q24. These areas are suggested as candidate regions for future targeted deoxyribonucleic acid (DNA) research in schizophrenia.
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PMID:Medical conditions in Ashkenazi schizophrenic pedigrees. 797 67

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