scholarly journals Optimization of a retroviral vector for transduction of human CD34 positive cells.

2006 ◽  
Vol 53 (4) ◽  
pp. 815-823 ◽  
Author(s):  
Anna Szyda ◽  
Maria Paprocka ◽  
Agnieszka Krawczenko ◽  
Katarzyna Lenart ◽  
Jerzy Heimrath ◽  
...  
Keyword(s):  
Cord Blood ◽  
Cell Lines ◽  
Ex Vivo ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Packaging Cell Line ◽  
Packaging Cell ◽  
Human Cd34 ◽  
Stem And Progenitor Cells

Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37 degrees C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.

scholarly journals Retroviral Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus

1999 ◽  
Vol 73 (7) ◽  
pp. 6114-6116 ◽  
Author(s):  
Hrvoje Miletic ◽  
Michael Bruns ◽  
Konstantinos Tsiakas ◽  
Birgit Vogt ◽  
Roya Rezai ◽  
...  
Keyword(s):  
Cell Lines ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Lymphocytic Choriomeningitis ◽  
Lymphocytic Choriomeningitis Virus ◽  
Transduction Efficiency ◽  
Murine Leukemia

ABSTRACT Pseudotyping can improve retroviral vector stability and transduction efficiency. Here, we describe a novel pseudotype of murine leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV). This pseudotype was stable during ultracentrifugation and infected several cell lines from different species. Moreover, LCMV glycoproteins were not cell toxic.

scholarly journals The Resistance of Retroviral Vectors Produced from Human Cells to Serum Inactivation In Vivo and In Vitro Is Primate Species Dependent

1999 ◽  
Vol 73 (8) ◽  
pp. 6708-6714 ◽  
Author(s):  
Nicholas J. DePolo ◽  
Cataline E. Harkleroad ◽  
Mordechai Bodner ◽  
Andrew T. Watt ◽  
Carol G. Anderson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Half Life ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Biologically Active ◽  
Primate Species ◽  
Phylogenetic Distance ◽  
Packaging Cell

ABSTRACT The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430–7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1,000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.

scholarly journals Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line

1998 ◽  
Vol 4 (3) ◽  
pp. 119-127
Author(s):  
Gerhard Bauer ◽  
Sybille Sauter ◽  
Carlos Ibanez ◽  
C.Robert Rice ◽  
Penelope Valdez ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cell Line ◽  
Progenitor Cells ◽  
Retroviral Vectors ◽  
Packaging Cell Line ◽  
Packaging Cell ◽  
Human Cd34

Ex vivo culture of human CD34+ cord blood cells with thrombopoietin (TPO) accelerates platelet engraftment in a NOD/SCID mouse model

2006 ◽  
Vol 34 (7) ◽  
pp. 943-950 ◽  
Author(s):  
Yvette van Hensbergen ◽  
Laurus F. Schipper ◽  
Anneke Brand ◽  
Manon C. Slot ◽  
Mick Welling ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cord Blood ◽  
Blood Cells ◽  
Scid Mouse ◽  
Ex Vivo ◽  
Human Cd34 ◽  
Scid Mouse Model ◽  
Cord Blood Cells ◽  
Ex Vivo Culture

scholarly journals Potent ex vivo expanded, human CD34+ cord blood-derived natural killer cells for cancer immunotherapy

2015 ◽  
Vol 3 (S2) ◽  
Author(s):  
Xiaokui Zhang ◽  
Lin Kang ◽  
Ivana Djuretic ◽  
Eric Law ◽  
Vanessa Voskinarian-Berse ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Cord Blood ◽  
Cancer Immunotherapy ◽  
Natural Killer ◽  
Ex Vivo ◽  
Killer Cells ◽  
Human Cd34

scholarly journals Improved transfer of the leukocyte integrin CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope

Blood ◽  
1995 ◽  
Vol 86 (6) ◽  
pp. 2379-2387 ◽  
Author(s):  
TR Jr Bauer ◽  
AD Miller ◽  
DD Hickstein
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Leukemia Virus ◽  
K562 Cells ◽  
Retroviral Vectors ◽  
Packaging Cell Line ◽  
Virus Receptor ◽  
Packaging Cell ◽  
Trna Primer ◽  
Gibbon Ape Leukemia Virus

Leukocyte adherence deficiency (LAD) is an inherited immunodeficiency disease caused by defects in the CD18 leukocyte integrin subunit. Transduction of CD18 into hematopoietic cells from children with LAD represents a potential therapy for this disorder. In an attempt to maximize transfer and expression of CD18, we evaluated retroviral vectors with and without the neomycin selectable marker, with a modified tRNA primer binding site designed to prevent inhibition of gene expression, and with two different viral envelope proteins produced by using the amphotropic retrovirus packaging cell line PA317 or the gibbon ape leukemia virus packaging cell line PG13. The vectors were tested using transducing K562/CD11b cells and LAD Epstein-Barr virus (EBV) B cells and measuring levels of cell-surface CD11/CD18 expression by fluorescence-activated cell sorter analysis. The best results were obtained with vectors made using PG13 packaging cells, for which about 25% of the K562 cells exposed once to the vectors expressed surface CD11b/CD18 and about 25% of the LAD EBV B cells exposed three times over a 3-day period to the vectors expressed surface CD11a/CD18. In contrast, transduction of cells under similar conditions with retroviral vectors produced using PA317 producer cells yielded less than 2% of the K562 cells and less than 4% of the LAD EBV B cells expressing the CD11/CD18 heterodimer on the cell surface. The presence or absence of the neomycin resistance gene or the modified tRNA primer had no effect on CD18 gene transfer rate or expression level. The increase in transduction with PG13 vectors correlated with Northern blotting and reverse transcription-polymerase chain reaction studies that indicated that both K562 cells and the LAD EBV B cells express transcripts for the gibbon ape leukemia virus receptor at higher levels than for the amphotropic virus receptor. These findings indicate that the transduction efficiency of retroviral packaging cell lines correlates with receptor gene expression in the target cells and that vectors made using PG13 cells may be efficacious for gene therapy for LAD and other diseases in which gene transfer to hematopoietic cells is required.

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line

2004 ◽  
Vol 6 (4) ◽  
pp. 374-386 ◽  
Author(s):  
François M. Lemoine ◽  
Mariana Mesel-Lemoine ◽  
Mustapha Cherai ◽  
Géraldine Gallot ◽  
Henri Vié ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cell Line ◽  
Retroviral Vector ◽  
Packaging Cell Line ◽  
Packaging Cell ◽  
Human T Lymphocytes ◽  
Selection Of

Adhesion to Fibronectin Maintains Regenerative Capacity During Ex Vivo Culture and Transduction of Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4612-4621 ◽  
Author(s):  
M.A. Dao ◽  
K. Hashino ◽  
I. Kato ◽  
J.A. Nolta
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Retroviral Vectors ◽  
Current Data ◽  
Regenerative Capacity ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Ex Vivo Culture

Abstract Recent reports have indicated that there is poor engraftment from hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo. However, inducing cells to cycle in culture is critical to the fields of ex vivo stem cell expansion and retroviral-mediated gene therapy. Through the use of a xenograft model, the current data shows that human hematopoietic stem and progenitor cells can traverse M phase ex vivo, integrate retroviral vectors, engraft, and sustain long-term hematopoiesis only if they have had the opportunity to engage their integrin receptors to fibronectin during the culture period. If cultured in suspension under the same conditions, transduction is undetectable and the long-term multilineage regenerative capacity of the primitive cells is severely diminished.

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