Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3710-3716 ◽  
Author(s):  
Peter A. Horn ◽  
Kirsten A. Keyser ◽  
Laura J. Peterson ◽  
Tobias Neff ◽  
Bobbie M. Thomasson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Lentiviral Vectors ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Lentiviral Transduction ◽  
Mobilized Peripheral Blood

Abstract The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34+ hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)– and granulocyte-colony stimulating factor (G-CSF)–primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

scholarly journals Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1482-1491 ◽  
Author(s):  
DM Bodine ◽  
NE Seidel ◽  
MS Gale ◽  
AW Nienhuis ◽  
D Orlic
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Stem Cell Factor ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Stimulating Factor

Abstract Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus- mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral- blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus- mediated gene transfer.

scholarly journals Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1482-1491 ◽  
Author(s):  
DM Bodine ◽  
NE Seidel ◽  
MS Gale ◽  
AW Nienhuis ◽  
D Orlic
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Stem Cell Factor ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Stimulating Factor

Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus- mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral- blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus- mediated gene transfer.

scholarly journals Enrichment of human hematopoietic stem cell activity in the CD34+Thy- 1+Lin- subpopulation from mobilized peripheral blood

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 368-378 ◽  
Author(s):  
L Murray ◽  
B Chen ◽  
A Galy ◽  
S Chen ◽  
R Tushinski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Peripheral Blood ◽  
Colony Stimulating Factor ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Stimulating Factor ◽  
Mobilized Peripheral Blood

Abstract The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. We have examined the presence of stem cells in mobilized peripheral blood (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34+Lineage- (Lin-) population. The kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34+Thy- 1+Lin- cell population, potentially containing PHSCs, was isolated by fluorescence activated cell sorting (FACS) and analyzed for activity. The multilineage differentiative capacity of this candidate stem cell- containing population in MPB was determined using an in vitro long-term culture system, in which cobblestone area formation was used as a means of detecting PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)-hu mice were implanted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area-forming cells (CAFC) after 7 weeks of culture was observed in a subpopulation of CD34+Lin- cells, which expressed low levels of Thy-1. This cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy-1+Lin- cell subset possessed a higher ability to engraft and to demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1+ and Thy-1- subpopulations were capable of donor T-cell engraftment at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-Term Follow-Up of Serially Transplanted Mice Receiving Extended Reduced Intensity Selection of MGMT-P140K Expressing Murine Repopulating Stem Cells Demonstrates Stable Oligo- to Polyclonal Hematopoiesis without Clonal Exhaustion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1286-1286
Author(s):  
Claudia Ball ◽  
Manfred Schmidt ◽  
Ingo Pilz ◽  
Monika Schrempp ◽  
Christof von Kalle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Reduced Intensity ◽  
Selection Of

Abstract In vivo selection of gene modified hematopoietic stem cells permanently increases the relative proportion of blood cells that carry a therapeutic transgene despite initially low gene transfer efficiency, thereby decreasing the likelihood of insertional mutagenesis and avoiding the need of myeloablative conditioning regimens. P140K Mutant O6-methylguanine-DNA methyltransferase (MGMT) enzyme confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as 1,3-bis-(2 chloroethyl)-1-nitrosourea (BCNU). We have previously shown that reduced intensity and toxicity BCNU/O6-BG selection allows efficient selection of MGMT-P140K expressing oligoclonal murine hematopoiesis. Nevertheless, whether long-term selection and the associated proliferative stress impairs long-term differentiation and proliferation of MGMT-P140K expressing stem cell clones is currently unknown and remains a major concern in the clinical application of MGMT selection. To address this question, serial transplantations of murine MGMT-P140K expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were done. After ex vivo gene transfer of an MGMT/IRES/eGFP encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and primary, secondary and tertiary recipient mice were subsequently treated every four weeks in order to exaggerate potential effects on long-term clonal behaviour. Lineage contribution of the transduced hematopoiesis was monitored by FACS over a total of 14 rounds of selection and clonality by LAM-PCR over a total of 12 rounds of selection. In primary mice the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without persisting peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential of transduced cells in 1st, 2nd and 3rd generation animals. LAM PCR analysis of peripheral blood samples revealed stable oligo- to polyclonal hematopoiesis in primary and secondary mice. Evidence for predominant clones or clonal exhaustion was not observed despite up to 12 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice that received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis, indicating extensive self-renewal of transplantable stem cells in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K expressing hematopoietic stem cells by repetitive chemotherapy does not affect their differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies for the amplification of a limited number of gene corrected clones in clinical gene therapy.

scholarly journals Foamy virus–mediated gene transfer to canine repopulating cells

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Hans-Peter Kiem ◽  
James Allen ◽  
Grant Trobridge ◽  
Erik Olson ◽  
Kirsten Keyser ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Blood Cells ◽  
Foamy Virus ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Cell Gene ◽  
Stem Cell Gene ◽  
Stem Cell Gene Therapy

AbstractFoamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)–expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/μL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP+, and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical.

Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2107-2107
Author(s):  
E.L.S. Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

Abstract A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo 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.

scholarly journals Enrichment of human hematopoietic stem cell activity in the CD34+Thy- 1+Lin- subpopulation from mobilized peripheral blood

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 368-378 ◽  
Author(s):  
L Murray ◽  
B Chen ◽  
A Galy ◽  
S Chen ◽  
R Tushinski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Peripheral Blood ◽  
Colony Stimulating Factor ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Stimulating Factor ◽  
Mobilized Peripheral Blood

The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. We have examined the presence of stem cells in mobilized peripheral blood (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34+Lineage- (Lin-) population. The kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34+Thy- 1+Lin- cell population, potentially containing PHSCs, was isolated by fluorescence activated cell sorting (FACS) and analyzed for activity. The multilineage differentiative capacity of this candidate stem cell- containing population in MPB was determined using an in vitro long-term culture system, in which cobblestone area formation was used as a means of detecting PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)-hu mice were implanted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area-forming cells (CAFC) after 7 weeks of culture was observed in a subpopulation of CD34+Lin- cells, which expressed low levels of Thy-1. This cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy-1+Lin- cell subset possessed a higher ability to engraft and to demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1+ and Thy-1- subpopulations were capable of donor T-cell engraftment at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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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