scholarly journals Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2579-2589 ◽  
Author(s):  
C Richardson ◽  
A Bank
Keyword(s):  
Gene Therapy ◽  
Drug Resistance ◽  
Human Gene ◽  
Multiple Drug Resistance ◽  
Fetal Liver ◽  
Multiple Drug ◽  
Hematopoietic Stem ◽  
Human Gene Therapy ◽  
Macrophage Cells

Abstract We have been using the human multiple drug resistance (MDR) gene to transduce murine hematopoietic cells via retroviruses as a model system for potential human gene therapy. In this paper, we show that transplantation of MDR-transduced midgestational fetal liver cells (FLCs) into lethally irradiated mice leads to the continued presence and expression of the human MDR gene in the short-lived granulocyte-macrophages of recipients' peripheral blood (PB) for up to 12 months. We have also shown the ability of this retroviral system to efficiently transduce several murine FLC subpopulations enriched for hematopoietic stem cells (FL-HSCs) both (1) short-term by MDR-polymerase chain reaction analysis of individual day 12 colony-forming unit-spleen and (2) long-term by in vivo maintenance of MDR and expression of its product, p-glycoprotein, up to 1 year in PB. More highly enriched FL-HSC subpopulations show the greatest number of circulating granulocyte-macrophage cells expressing MDR long-term. These studies also show that preselection by fluorescence-activated cell sorting of MDR-transduced and -expressing cells before transplant significantly increases the percentage of circulating granulocyte-macrophage cells that express MDR at all time points analyzed posttransplant as compared with unsorted cells transduced in the same manner (P < .01). These results have potentially significant implications for future human gene therapy trials.

scholarly journals Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2579-2589 ◽  
Author(s):  
C Richardson ◽  
A Bank
Keyword(s):  
Gene Therapy ◽  
Drug Resistance ◽  
Human Gene ◽  
Multiple Drug Resistance ◽  
Fetal Liver ◽  
Multiple Drug ◽  
Hematopoietic Stem ◽  
Human Gene Therapy ◽  
Macrophage Cells

We have been using the human multiple drug resistance (MDR) gene to transduce murine hematopoietic cells via retroviruses as a model system for potential human gene therapy. In this paper, we show that transplantation of MDR-transduced midgestational fetal liver cells (FLCs) into lethally irradiated mice leads to the continued presence and expression of the human MDR gene in the short-lived granulocyte-macrophages of recipients' peripheral blood (PB) for up to 12 months. We have also shown the ability of this retroviral system to efficiently transduce several murine FLC subpopulations enriched for hematopoietic stem cells (FL-HSCs) both (1) short-term by MDR-polymerase chain reaction analysis of individual day 12 colony-forming unit-spleen and (2) long-term by in vivo maintenance of MDR and expression of its product, p-glycoprotein, up to 1 year in PB. More highly enriched FL-HSC subpopulations show the greatest number of circulating granulocyte-macrophage cells expressing MDR long-term. These studies also show that preselection by fluorescence-activated cell sorting of MDR-transduced and -expressing cells before transplant significantly increases the percentage of circulating granulocyte-macrophage cells that express MDR at all time points analyzed posttransplant as compared with unsorted cells transduced in the same manner (P < .01). These results have potentially significant implications for future human gene therapy trials.

Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

1995 ◽  
Vol 18 (1-2) ◽  
pp. 119-124
Author(s):  
Arthur Bank ◽  
Maureen Ward ◽  
Christine Richardson ◽  
Patricia Pioli ◽  
Charles Hesdorffer
Keyword(s):  
Gene Therapy ◽  
Drug Resistance ◽  
Resistance Gene ◽  
Human Gene ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
Human Gene Therapy ◽  
Drug Resistance Gene ◽  
Multiple Drug Resistance Gene

scholarly journals Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 855-862 ◽  
Author(s):  
T Moritz ◽  
P Dutt ◽  
X Xiao ◽  
D Carstanjen ◽  
T Vik ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Progenitor Cells ◽  
Human Gene ◽  
Virus Titer ◽  
Retroviral Vectors ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Human Gene Therapy ◽  
Carboxy Terminal

Abstract Efficient transduction of reconstituting hematopoletic stem cells (HSC) is currently only possible by cocultivation of target cells directly on producer cell lines, a method not applicable to human gene therapy protocols. Our laboratory has previously shown adhesion of primitive hematopoletic stem and progenitor cells to the carboxy-terminal 30/35- kD fragment of the extracellular matrix molecule fibronectin (FN 30/35) (Nature 352:438, 1991) and increased transduction of human hematopoietic progenitor cells via retroviral vectors while adherent to this fragment (J Clin Invest 93:1451, 1994). Here we report that (1) transduction of reconstituting murine HSC assayed 12 months after infection with retrovirus supernatant on FN 30/35 is as effective as cocultivation directly on producer cells; (2) recombinant retrovirus particles directly adhere to FN 30/35 in a quantitative and dose- dependent fashion; and (3) increased transduction efficiency on FN 30/ 35 does not appear to be associated with increased cell proliferation or activation of protein phosphorylation typically induced by integrin- fibronectin interactions. Therefore, we speculate that supernatant infection of HSC on FN 30/35 leads to colocalization of retrovirus particles and target cells on FN 30/35 molecule with a large increase in local virus titer presented to the cell. These findings have direct and important implications for the modification of current human gene therapy protocols.

Gene therapy of apolipoprotein E–deficient mice using a novel macrophage-specific retroviral vector

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 485-491 ◽  
Author(s):  
Peter J. Gough ◽  
Elaine W. Raines
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Therapy ◽  
Apolipoprotein E ◽  
Human Gene ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Hematopoietic Stem ◽  
Human Gene Therapy ◽  
Deficient Mice

The use of retroviral gene transfer into hematopoietic stem cells for human gene therapy has been hampered by the absence of retroviral vectors that can generate long-lasting, lineage-specific gene expression. We developed self-inactivating retroviral vectors that incorporate gene-regulatory elements from the macrophage-restricted human CD68 gene. Through the transplantation of transduced murine hematopoietic stem cells (HSCs), we show that a vector incorporating a 342–base pair (bp) fragment of 5′ flanking sequence from the CD68 gene, in addition to the CD68 first intron, was able to direct macrophage-specific expression of an enhanced green fluorescent protein (EGFP) reporter gene in inflammatory cell exudates and lymphoid organs in vivo. Levels of EGFP expression generated by this vector were greater than those generated by a standard Moloney murine leukemia retroviral vector, and they were stable for at least a year after transplantation of transduced HSCs. To evaluate the ability of this vector to generate therapeutically useful levels of gene expression, we transplanted apolipoprotein E (ApoE)–deficient HSCs transduced with a virus encoding ApoE into ApoE-deficient mice. Macrophages from these mice expressed levels of ApoE that were comparable to those from wild-type mice, and vector-driven expression of ApoE in macrophages was sufficient to reverse both hypercholesterolemia and atherosclerotic lesion development. The future application of this retroviral vector should provide a powerful tool to further elucidate macrophage function and for human gene therapy.

scholarly journals High-Efficiency Gene Transfer into Normal and Adenosine Deaminase-Deficient T Lymphocytes Is Mediated by Transduction on Recombinant Fibronectin Fragments

1998 ◽  
Vol 72 (6) ◽  
pp. 4882-4892 ◽  
Author(s):  
Karen E. Pollok ◽  
Helmut Hanenberg ◽  
Timothy W. Noblitt ◽  
Wendy L. Schroeder ◽  
Ikunoshin Kato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
T Lymphocytes ◽  
Adenosine Deaminase ◽  
High Efficiency ◽  
Human Gene ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Human Gene Therapy ◽  
Human T Lymphocytes

ABSTRACT Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.

Bone Marrow as a Source of Hematopoietic Stem Cells for Human Gene Therapy of β-Thalassemia

2011 ◽  
Vol 22 (4) ◽  
pp. 507-513 ◽  
Author(s):  
Marta Claudia Frittoli ◽  
Erika Biral ◽  
Barbara Cappelli ◽  
Matilde Zambelli ◽  
Maria Grazia Roncarolo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Human Gene ◽  
Hematopoietic Stem ◽  
Human Gene Therapy

Gene Therapy 2000

Hematology ◽  
2000 ◽  
Vol 2000 (1) ◽  
pp. 376-393 ◽  
Author(s):  
David A. Williams ◽  
Arthur W. Nienhuis ◽  
Robert G. Hawley ◽  
Franklin O. Smith
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Human Gene ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Regulatory Issues ◽  
Human Gene Therapy

Abstract This article reviews 1) the use of gene transfer methods to genetically manipulate hematopoietic stem cell targets, 2) recent advances in technology that are addressing problems that have prevented widespread successful translation of gene transfer approaches for the cure of disease, and 3) recent regulatory issues related to human gene therapy trials. In Section I, Dr. Nienhuis describes the use of alternative viral envelopes and vector systems to improve efficiency of transduction of hematopoietic stem cells. Major limitations of stem cell transduction are related to low levels of viral receptors on the stem cells of large animal species and the low frequency of cycling stem cells in the bone marrow. Attempts to circumvent these limitations by exploiting non-oncoretroviral vectors and pseudotyping of Moloney vectors with alternative envelopes are discussed. In Section II, Dr. Hawley addresses new strategies to improve the expression of transgenes in cells derived from long-term reconstituting hematopoietic stem cells. Transgene silencing in transduced hematopoietic stem cells remains an obstacle to gene therapy for some gene sequences. New generations of retroviral backbones designed to both improve expression and reduce silencing in primary cells are explored. In Section III, Drs. Smith and Cornetta update regulatory issues related to human gene therapy trials. Increased scrutiny of human trials has led to changes in requirements and shifts in emphasis of existing regulations, which apply to human gene therapy trials. The current Food and Drug Administration's structure and regulations and the roles of the Recombinant DNA Advisory Committee of the NIH and other sponsors and partners in gene therapy trials are reviewed.

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