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Query: UMLS:C0038362 (stomatitis)
 8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.
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PMID:High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells. 899 65

Hematopoiesis is a balance between proliferation and differentiation that may be modulated by environmental signals. Notch receptors and their ligands are highly conserved during evolution and have been shown to regulate cell fate decisions in multiple developmental systems. To assess whether Notch1 signaling may regulate human hematopoiesis to maintain cells in an immature state, we transduced a vesicular stomatitis virus G-protein (VSV-G) pseudo-typed bicistronic murine stem cell virus (MSCV)-based retroviral vector expressing a constitutively active form of Notch1 (ICN) and green fluorescence protein into the differentiation competent HL-60 cell line and primary cord blood-derived CD34(+) cells. In addition, we observed endogenous Notch1 expression on the surface of both HL-60 cells and primary CD34(+) cells, and therefore exposed cells to Notch ligand Jagged2, expressed on NIH3T3 cells. Both ligand-independent and ligand-dependent activation of Notch resulted in delayed acquisition of differentiation markers by HL-60 cells and cord blood CD34(+) cells. In addition, primary CD34(+) cells retained their ability to form immature colonies, colony-forming unit-mix (CFU-mix), whereas control cells lost this capacity. Activation of Notch1 correlated with a decrease in the fraction of HL-60 cells that were in G0/G1 phase before acquisition of a mature cell phenotype. This enhanced progression through G1 was noted despite preservation of the proliferative rate of the cells and the overall length of the cell cycle. These findings show that Notch1 activation delays human hematopoietic differentiation and suggest a link of Notch differentiation effects with altered cell cycle kinetics.
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PMID:Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics. 992 Aug 32

Lentivirus vectors based on human immunodeficiency virus (HIV) type 1 (HIV-1) constitute a recent development in the field of gene therapy. A key property of HIV-1-derived vectors is their ability to infect nondividing cells. Although high-titer HIV-1-derived vectors have been produced, concerns regarding safety still exist. Safety concerns arise mainly from the possibility of recombination between transfer and packaging vectors, which may give rise to replication-competent viruses with pathogenic potential. We describe a novel lentivirus vector which is based on HIV, simian immunodeficiency virus (SIV), and vesicular stomatitis virus (VSV) and which we refer to as HIV/SIVpack/G. In this system, an HIV-1-derived genome is encapsidated by SIVmac core particles. These core particles are pseudotyped with VSV glycoprotein G. Because the nucleotide homology between HIV-1 and SIVmac is low, the likelihood of recombination between vector elements should be reduced. In addition, the packaging construct (SIVpack) for this lentivirus system was derived from SIVmac1A11, a nonvirulent SIV strain. Thus, the potential for pathogenicity with this vector system is minimal. The transduction ability of HIV/SIVpack/G was demonstrated with immortalized human lymphocytes, human primary macrophages, human bone marrow-derived CD34(+) cells, and primary mouse neurons. To our knowledge, these experiments constitute the first demonstration that the HIV-1-derived genome can be packaged by an SIVmac capsid. We demonstrate that the lentivirus vector described here recapitulates the biological properties of HIV-1-derived vectors, although with increased potential for safety in humans.
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PMID:Lentivirus vectors using human and simian immunodeficiency virus elements. 1007 31

Lentiviral vectors have been proposed as a more efficient alternative to Moloney murine leukemia virus-based retroviral vectors for transduction of human hematopoietic progenitors and stem cells. These studies were designed to evaluate the conditions that influence transduction frequency of CD34(+) progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34(+) human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carrying an internal human cytomegalovirus promoter driving enhanced green fluorescent protein (eGFP) expression. Using fluorescence-activated cell sorting analysis of eGFP, we observed pseudotransduction beginning at the time of vector addition and lasting up to 24 h in CD34(+) cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP expression for up to 10 days in CD34(+) cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34(+) cells on fibronectin-coated plates. Transduction frequency was related directly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transduced CD34(+) cells equally, demonstrating a lack of dependence on HIV-1 accessory proteins. These findings will be useful for the optimal utilization of this new class of vectors for transduction of human hematopoietic stem cells.
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PMID:Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors. 1089 30

Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5' cytomegalovirus early promoter, the 5' viral sequences up to the 5' end of gag required for reverse transcription and packaging, the Rev-responsive element, a gene-expressing cassette, and the 3' long terminal repeat (LTR). Production of SIV vector particles was achieved by transfecting 293T cells with the vector DNA and helper constructs coding for the viral genes and the vesicular stomatitis virus glycoprotein G envelope. These SIV vectors were found to have transducing titers reaching 10(7) transducing units/ml on HeLa cells and to deliver a gene without transfer of helper functions to target cells. The central polypurine tract can be included in the minimal vector, resulting in a two- to threefold increase in the transduction titers on dividing or growth-arrested cells. Based on this minimal SIV vector, a sin vector was designed by deleting 151 nucleotides in the 3' LTR U3 region, and this SIV sin vector retained high transduction titers. Furthermore, the minimal SIV vector was efficient at transducing terminally differentiated human CD34(+) cell-derived or monocyte-derived dendritic cells (DCs). Results show that up to 40% of human primary DCs can be transduced by the SIV vectors. This opens a new perspective in the field of immunotherapy.
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PMID:Development of minimal lentivirus vectors derived from simian immunodeficiency virus (SIVmac251) and their use for gene transfer into human dendritic cells. 1095 29

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34(+) cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin(-) bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.
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PMID:Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to moloney murine leukemia virus-based vectors. 1107 32

The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34(+) cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34(+) population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400,000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells. (Blood. 2000;96:3725-3733)
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PMID:Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells. 1109 53

The methods available to efficiently transduce human CD34(+) hematopoietic stem cells (HSCs) derived from mobilized peripheral blood, such that they fully retain their engraftment potential and maintain high levels of transgene expression in vivo, have been unsatisfactory. The current murine retrovirus-based gene transfer systems require dividing cells for efficient transduction, and therefore the target HSCs must be activated ex vivo by cytokines to cycle, which may limit their engrafting ability. Lentivirus-based gene transfer systems do not require cell division and, thus, may allow for efficient gene transfer to human HSCs in the absence of any ex vivo cytokine stimulation. We constructed human immunodeficiency virus (HIV)-based vectors and compared them in vitro and in vivo with MuLV-based vectors in their ability to transduce unstimulated human CD34(+) HSCs isolated from mobilized peripheral blood. Both sets of vectors contained the marker gene that expresses the enhanced green fluorescent protein (EGFP) for evaluating transduction efficiency and were pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or the amphotropic murine leukemia virus envelope (A-MULV Env). The VSV-G-pseudotyped HIV-based vectors containing an internal mouse phosphoglycerate kinase promoter (PGK) were able to transduce up to 48% of the unstimulated CD34(+) cells as measured by EGFP expression. When these cells were injected into the human fetal thymus implants of irradiated SCID-hu Thy/Liv mice, up to 18% expressed EGFP after 8 weeks in vivo. In contrast, the MULV-based vectors were effective at transducing HSCs only in the presence of cytokines. Our results demonstrate that the improved HIV-based gene transfer system can effectively transduce unstimulated human CD34(+) HSCs, which can then differentiate into thymocytes and provide long-term transgene expression in vivo.
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PMID:Efficient human immunodeficiency virus-based vector transduction of unstimulated human mobilized peripheral blood CD34+ cells in the SCID-hu Thy/Liv model of human T cell lymphopoiesis. 1124 32

Both oncoretroviral and lentiviral vectors have been shown to transduce CD34(+) human hematopoietic stem cells (HSC) capable of establishing human hematopoiesis in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice that support partially human hematopoiesis. We and others have reported that murine stem cell virus (MSCV)-based oncoretroviral vectors efficiently transduced HSC that had been cultured ex vivo for 4-7 days with cytokines, resulting in transgene expression in lymphoid and myeloid progenies of SCID-engrafting cells 4-8 weeks post-transplantation. Although lentiviral vectors have been demonstrated to transduce HSC under minimal ex vivo culture conditions, concerns exist regarding the level of transgene expression mediated by these vectors. We therefore evaluated a novel hybrid lentiviral vector (GIN-MU3), in which the U3 region of the HIV-1 long terminal repeat was replaced by the MSCV U3 region (MU3). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G envelope protein-pseudotyped lentiviruses during a 48-hour culture period. After a total of 4 days in culture, transduced cells were transplanted into NOD/SCID mice to examine gene transfer and expression in engrafting human cells. Fifteen weeks post-transplantation, 37% +/- 12% of engrafted human cells expressed the green fluorescence protein (GFP) gene introduced by the lentiviral vector. High levels of GFP expression were observed in lymphoid, myeloid and erythroid progenies, and in engrafted human cells that retained the CD34(+) phenotype 15 weeks post-transplantation. This study provides evidence that lentiviral vectors transduced both short-term and long-term engrafting human cells, and mediated persistent transgene expression at high levels in multiple lineages of hematopoietic cells.
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PMID:High levels of transgene expression following transduction of long-term NOD/SCID-repopulating human cells with a modified lentiviral vector. 1135 50

Forty-one patients with multiple myeloma were treated with a novel stem cell mobilisation regimen. The primary end points were adequate stem cell mobilising ability (>1% circulating CD34-positive cells) and collection (> or = 4 x 10(6) CD34-positive cells/kg), and safety. The secondary end point was activity against myeloma. The regimen (d-TEC) consisted of dexamethasone, paclitaxel 200 mg/m(2) i.v., etoposide 60 mg/kg i.v., cyclophosphamide 3 g/m(2) i.v., and G-CSF 5-10 microg/kg/day i.v. A total of 84 cycles were administered to these 41 individuals. Patient characteristics included a median age of 53 years, a median of five prior chemotherapy cycles, and a median interval of 10 months from diagnosis of myeloma to first cycle of d-TEC. Seventy-five percent of the patients had stage II or III disease, 50% had received carmustine and/or melphalan previously, and 25% had received prior radiation therapy. Eighty-eight percent of patients mobilised adequately after the first cycle of d-TEC and 91% mobilized adequately after the second cycle. An adequate number of stem cells were collected in 32 patients. Of the remaining nine patients, three mobilised, but stem cells were not collected, two mobilised but stem cell collection was < 4 x 10(6) CD34-positive cells/kg, three did not mobilise, and one died of disease progression. Major toxicities included pancytopenia, alopecia, fever and stomatitis. One patient died from multi-organ failure and progressive disease. Fifty percent of evaluable patients demonstrated a partial response and 28.6% of patients had a minor response. This novel dose-intense regimen was safe, capable of stem cell mobilisation and collection, even in heavily pre-treated patients, and active against the underlying myeloma.
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PMID:Dexamethasone, paclitaxel, etoposide, cyclophosphamide (d-TEC) and G-CSF for stem cell mobilisation in multiple myeloma. 1150 31


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