scholarly journals 396. Factors Affecting Retroviral Gene Transfer to Human CD34+ Cells

2004 ◽  
Vol 9 ◽  
pp. S152
Keyword(s):  
Gene Transfer ◽  
Factors Affecting ◽  
Retroviral Gene Transfer ◽  
Human Cd34 ◽  
Cd34 Cells

scholarly journals Characterization of Retroviral Gene Transfer into Highly Purified Human CD34− Cells with Primitive Hematopoietic Capacity

2002 ◽  
Vol 5 (5) ◽  
pp. 635-643 ◽  
Author(s):  
Barbara Murdoch ◽  
Lisa Gallacher ◽  
Kristin Chadwick ◽  
Mickie Bhatia
Keyword(s):  
Gene Transfer ◽  
Retroviral Gene Transfer ◽  
Human Cd34 ◽  
Cd34 Cells

Factors affecting retrovirus-mediated gene transfer to human CD34+ cells

2004 ◽  
Vol 6 (7) ◽  
pp. 724-733 ◽  
Author(s):  
Youngtae Hong ◽  
Karim Lee ◽  
Seung Shin Yu ◽  
Sujeong Kim ◽  
Joong-Gon Kim ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factors Affecting ◽  
Human Cd34 ◽  
Cd34 Cells

Optimization of Fibronectin-Assisted Retroviral Gene Transfer into Human CD34+Hematopoietic Cells

1997 ◽  
Vol 8 (18) ◽  
pp. 2193-2206 ◽  
Author(s):  
Helmut Hanenberg ◽  
Kimikazu Hashino ◽  
Haruko Konishi ◽  
Randy A. Hock ◽  
Ikunoshin Kato ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Hematopoietic Cells ◽  
Retroviral Gene Transfer ◽  
Human Cd34

Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4622-4622
Author(s):  
Nadja Grund ◽  
Patrick Maier ◽  
Uwe Appelt ◽  
Heike Allgayer ◽  
Frederik Wenz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
In Silico ◽  
Integration Site ◽  
General Distribution ◽  
Cytostatic Drug ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.

scholarly journals Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3290-3297 ◽  
Author(s):  
GM Crooks ◽  
DB Kohn
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Gene Transfer ◽  
Hematopoietic Cells ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Virus Binding ◽  
Steel Factor ◽  
Human Cd34 ◽  
Cd34 Cells

Gene transfer into human cells using murine amphotropic retroviral vectors is the basic technique used in most current gene therapy studies. The identity of the cell surface receptor for the amphotropic envelope remains unknown and thus its importance in gene transfer is poorly understood. We have measured specific retrovirus binding to cells to study amphotropic virus receptor regulation in human CD34+ bone marrow (BM) progenitors and primitive CD34+CD38- human hematopoietic cells. The rat monoclonal antibody 83A25 recognizes an epitope common to the envelope glycoprotein of all classes of Moloney murine leukemia virus. Indirect fluorescent labeling of 83A25 allows flow cytometric analysis of specific virus-cell interactions and is an indirect measure of specific receptors. Using this assay, amphotropic virus binding to fresh CD34+ cells was minimal. However, when CD34+ cells were cultured with or without growth factors for 4 days, specific binding of amphotropic retrovirus was readily shown. Inclusion of interleukin-3 (IL-3), IL-6, and Steel factor in cultures increased the fluorescence associated with amphotropic virus binding by twofold to four-fold (mean fold increase 2.7 +/- 0.84). Virus binding to CD34+CD38- cells was shown only in those cells culture in IL-3, IL-6, and Steel factor. These results suggest that certain cytokines may cause an increase in the number and/or affinity of amphotropic receptors on primitive human hematopoietic cells. Upregulation of viral receptor expression may be one of the mechanisms by which cytokines enhance gene transfer into primitive BM cells.

scholarly journals Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3290-3297 ◽  
Author(s):  
GM Crooks ◽  
DB Kohn
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Gene Transfer ◽  
Hematopoietic Cells ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Virus Binding ◽  
Steel Factor ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Gene transfer into human cells using murine amphotropic retroviral vectors is the basic technique used in most current gene therapy studies. The identity of the cell surface receptor for the amphotropic envelope remains unknown and thus its importance in gene transfer is poorly understood. We have measured specific retrovirus binding to cells to study amphotropic virus receptor regulation in human CD34+ bone marrow (BM) progenitors and primitive CD34+CD38- human hematopoietic cells. The rat monoclonal antibody 83A25 recognizes an epitope common to the envelope glycoprotein of all classes of Moloney murine leukemia virus. Indirect fluorescent labeling of 83A25 allows flow cytometric analysis of specific virus-cell interactions and is an indirect measure of specific receptors. Using this assay, amphotropic virus binding to fresh CD34+ cells was minimal. However, when CD34+ cells were cultured with or without growth factors for 4 days, specific binding of amphotropic retrovirus was readily shown. Inclusion of interleukin-3 (IL-3), IL-6, and Steel factor in cultures increased the fluorescence associated with amphotropic virus binding by twofold to four-fold (mean fold increase 2.7 +/- 0.84). Virus binding to CD34+CD38- cells was shown only in those cells culture in IL-3, IL-6, and Steel factor. These results suggest that certain cytokines may cause an increase in the number and/or affinity of amphotropic receptors on primitive human hematopoietic cells. Upregulation of viral receptor expression may be one of the mechanisms by which cytokines enhance gene transfer into primitive BM cells.

Optimization of the Sleeping Beauty Transposon System to Achieve Stable Transgene Expression in Human CD34+ Hematopoietic Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3527-3527
Author(s):  
Teiko Sumiyoshi ◽  
Roger P Hollis ◽  
Nathalia Holt ◽  
Donald B. Kohn
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Sleeping Beauty ◽  
Egfp Expression ◽  
Stable Gene ◽  
Human Cd34 ◽  
Gfp Reporter ◽  
Cd34 Cells

Abstract Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. Transposon-mediated gene integration may have advantages over viral vectors, with a greater transgene carrying capacity and potentially safer integration site profile. Due to these characteristics of SB, there has been great interest in its potential use in hematopoietic stem cell (HSC) gene therapy. In this study, we optimized the SB transposon-mediated gene transfer system to achieve higher stable transgene expression in K562 human erythroleukemia cells, Jurkat human T-lymphoid cells, and primary human CD34+ hematopoietic progenitor cells. The SB transposon system was optimized by two approaches: to increase the transposition efficacy, a hyperactive mutant of SB, HSB16, was used (Baus et al.; Mol Ther12:1148, 2005); to optimize the expression of the SB transposase and the transgene cassette carried by the transposon, three different viral and cellular promoters were evaluated, including the modified MPSV long terminal repeat (MNDU3) enhancer-promoter, the human cytomegalovirus (hCMV) immediate-early region enhancer-promoter, and the human elongation factor 1 (hEF1a) promoter. SB components were delivered in trans into the target cells by nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression using fluorescent-activated cell sorting (FACS) analysis over 3–4 weeks. The functional assay showed that HSB16 was a more efficient enzyme compared to the original SB. In purified human cord blood CD34+ cells, HSB16 achieved nearly 7-fold higher long-term transgene expression with 90% less plasmid DNA (from 10 mcg of SB reduced to 1 mcg of HSB16) than the original SB transposase. The highest level of stable transgene integration in all three cell types was achieved using the hEF1a promoter to express HSB16 in comparison to either the hCMV or MND promoter. Our data also suggested that optimal GFP reporter gene expression from the integrated transposon was influenced by the type of promoter and the target cell type. Significantly higher levels of eGFP expression (5-fold) were achieved with the hEF1a promoter in Jurkat human T cells, compared to that achieved with the MND promoter; in contrast the MND promoter expressed GFP at the highest level in K562 myeloid cells. In primary human CD34+ cord blood progenitors, optimal transgene integration and expression was achieved using the hEF1a promoter to express the SB transposase combined with the MND promoter to express GFP reporter, when studied under conditions directing myeloid differentiation. Stable transgene expression was achieved at levels up to 27% for over 4 weeks after optimized gene transfer to CD34+ cells (ave=17%, n=4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34+ progenitor cells are currently in progress. In conclusion, the optimized SB transposon system in primary human CD34+ hematopoietic progenitors reported here has improved the stable gene transfer efficiency by 29-fold, compared to our prior published data (< 1% - Hollis et al.; Exp Hematol34:1333, 2006). The long-term stable gene expression achieved by our optimized SB transposon system shows promise for further advancement of non-viral based HSC gene therapy.

scholarly journals Overexpression of Gibbon Ape Leukemia Virus (GALV) Receptor (GLVR1) on Human CD34+ Cells Increases Gene Transfer Mediated by GALV Pseudotyped Vectors

2002 ◽  
Vol 6 (3) ◽  
pp. 400-406 ◽  
Author(s):  
Thomas Relander ◽  
Ann C.M. Brun ◽  
Karin Olsson ◽  
Lene Pedersen ◽  
Johan Richter
Keyword(s):  
Gene Transfer ◽  
Leukemia Virus ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Gibbon Ape Leukemia Virus

scholarly journals Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

Gene Therapy ◽  
2003 ◽  
Vol 10 (12) ◽  
pp. 1041-1048 ◽  
Author(s):  
F Sakurai ◽  
H Mizuguchi ◽  
T Hayakawa
Keyword(s):  
Gene Transfer ◽  
Adenovirus Type ◽  
Human Cd34 ◽  
Efficient Gene ◽  
Cd34 Cells
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