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Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

CE Dunbar, M Cottler-Fox, JA O'Shaughnessy, S Doren, C Carter, R Berenson, S Brown, RC Moen, J Greenblatt and FM Stewart

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1652, USA.

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady- state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.

Volume 85, Issue 11, pp. 3048-3057, 06/01/1995
Copyright © 1995 by The American Society of Hematology


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J. C.M. van der Loo, H. Hanenberg, R. J. Cooper, F.-Y. Luo, E. N. Lazaridis, and D. A. Williams
Nonobese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) Mouse as a Model System to Study the Engraftment and Mobilization of Human Peripheral Blood Stem Cells
Blood, October 1, 1998; 92(7): 2556 - 2570.
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J.F. Tisdale, Y. Hanazono, S.E. Sellers, B.A. Agricola, M.E. Metzger, R.E. Donahue, and C.E. Dunbar
Ex Vivo Expansion of Genetically Marked Rhesus Peripheral Blood Progenitor Cells Results in Diminished Long-Term Repopulating Ability
Blood, August 15, 1998; 92(4): 1131 - 1141.
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K. E. Pollok, H. Hanenberg, T. W. Noblitt, W. L. Schroeder, I. Kato, D. Emanuel, and D. A. Williams
High-Efficiency Gene Transfer into Normal and Adenosine Deaminase-Deficient T Lymphocytes Is Mediated by Transduction on Recombinant Fibronectin Fragments
J. Virol., June 1, 1998; 72(6): 4882 - 4892.
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P. Veena, C. M. Traycoff, D. A. Williams, J. McMahel, S. Rice, K. Cornetta, and E. F. Srour
Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes
Blood, May 15, 1998; 91(10): 3693 - 3701.
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D. Orlic, L. J. Girard, S. M. Anderson, L. C. Pyle, M. C. Yoder, H. E. Broxmeyer, and D. M. Bodine
Identification of Human and Mouse Hematopoietic Stem Cell Populations Expressing High Levels of mRNA Encoding Retrovirus Receptors
Blood, May 1, 1998; 91(9): 3247 - 3254.
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A. Rambaldi, G. Borleri, G. Dotti, P. Bellavita, R. Amaru, A. Biondi, and T. Barbui
Innovative Two-Step Negative Selection of Granulocyte Colony-Stimulating Factor-Mobilized Circulating Progenitor Cells: Adequacy for Autologous and Allogeneic Transplantation
Blood, March 15, 1998; 91(6): 2189 - 2196.
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J. Virol.Home page
M. Onodera, D. M. Nelson, A. Yachie, G. J. Jagadeesh, B. A. Bunnell, R. A. Morgan, and R. M. Blaese
Development of Improved Adenosine Deaminase Retroviral Vectors
J. Virol., March 1, 1998; 72(3): 1769 - 1774.
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A. K. Fielding, M. Maurice, F. J. Morling, F.-L. Cosset, and S. J. Russell
Inverse Targeting of Retroviral Vectors: Selective Gene Transfer in a Mixed Population of Hematopoietic and Nonhematopoietic Cells
Blood, March 1, 1998; 91(5): 1802 - 1809.
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S. D. Lyman and S. E. W. Jacobsen
c-kit Ligand and Flt3 Ligand: Stem/Progenitor Cell Factors With Overlapping Yet Distinct Activities
Blood, February 15, 1998; 91(4): 1101 - 1134.
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J. Virol.Home page
D. von Laer, S. Thomsen, B. Vogt, M. Donath, J. Kruppa, A. Rein, W. Ostertag, and C. Stocking
Entry of Amphotropic and 10A1 Pseudotyped Murine Retroviruses Is Restricted in Hematopoietic Stem Cell Lines
J. Virol., February 1, 1998; 72(2): 1424 - 1430.
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L. Jin, H. Asano, and C. Anthony Blau
Stimulating Cell Proliferation Through the Pharmacologic Activation of c-kit
Blood, February 1, 1998; 91(3): 890 - 897.
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B. Verhasselt, M. De Smedt, R. Verhelst, E. Naessens, and J. Plum
Retrovirally Transduced CD34++ Human Cord Blood Cells Generate T Cells Expressing High Levels of the Retroviral Encoded Green Fluorescent Protein Marker In Vitro
Blood, January 15, 1998; 91(2): 431 - 440.
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M. Rosenzweig, D. F. Marks, D. Hempel, M. Heusch, G. Kraus, F. Wong-Staal, and R. P. Johnson
Intracellular Immunization of Rhesus CD34+ Hematopoietic Progenitor Cells With a Hairpin Ribozyme Protects T Cells and Macrophages From Simian Immunodeficiency Virus Infection
Blood, December 15, 1997; 90(12): 4822 - 4831.
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H.-P. Kiem, S. Heyward, A. Winkler, J. Potter, J. M. Allen, A. D. Miller, and R. G. Andrews
Gene Transfer into Marrow Repopulating Cells: Comparison Between Amphotropic and Gibbon Ape Leukemia Virus Pseudotyped Retroviral Vectors in a Competitive Repopulation Assay in Baboons
Blood, December 1, 1997; 90(11): 4638 - 4645.
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M. F.A. Bierhuizen, Y. Westerman, T. P. Visser, W. Dimjati, A. W. Wognum, and G. Wagemaker
Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells
Blood, November 1, 1997; 90(9): 3304 - 3315.
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L. Humeau, C. Chabannon, M. T. Firpo, P. Mannoni, C. Bagnis, M.-G. Roncarolo, and R. Namikawa
Successful Reconstitution of Human Hematopoiesis in the SCID-hu Mouse by Genetically Modified, Highly Enriched Progenitors Isolated From Fetal Liver
Blood, November 1, 1997; 90(9): 3496 - 3506.
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Proc. Natl. Acad. Sci. USAHome page
H. L. Malech, P. B. Maples, N. Whiting-Theobald, G. F. Linton, S. Sekhsaria, S. J. Vowells, F. Li, J. A. Miller, E. DeCarlo, S. M. Holland, et al.
Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease
PNAS, October 28, 1997; 94(22): 12133 - 12138.
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Proc. Natl. Acad. Sci. USAHome page
E. Conneally, J. Cashman, A. Petzer, and C. Eaves
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice
PNAS, September 2, 1997; 94(18): 9836 - 9841.
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D. A. Persons, J. A. Allay, E. R. Allay, R. J. Smeyne, R. A. Ashmun, B. P. Sorrentino, and A. W. Nienhuis
Retroviral-Mediated Transfer of the Green Fluorescent Protein Gene Into Murine Hematopoietic Cells Facilitates Scoring and Selection of Transduced Progenitors In Vitro and Identification of Genetically Modified Cells In Vivo
Blood, September 1, 1997; 90(5): 1777 - 1786.
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E. L.W. Kittler, S. O. Peters, R. B. Crittenden, M. E. Debatis, H. S. Ramshaw, F. M. Stewart, and P. J. Quesenberry
Cytokine-Facilitated Transduction Leads to Low-Level Engraftment in Nonablated Hosts
Blood, July 15, 1997; 90(2): 865 - 872.
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I. Rappold, B. L. Ziegler, I. Kohler, S. Marchetto, O. Rosnet, D. Birnbaum, P. J. Simmons, A. C.W. Zannettino, B. Hill, S. Neu, et al.
Functional and Phenotypic Characterization of Cord Blood and Bone Marrow Subsets Expressing FLT3 (CD135) Receptor Tyrosine Kinase
Blood, July 1, 1997; 90(1): 111 - 125.
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The OncologistHome page
C. Bagnis and P. Mannoni
Stem Cell-Based Gene Therapy
Oncologist, June 1, 1997; 2(3): 196 - 202.
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R.V.B. Emmons, S. Doren, J. Zujewski, M. Cottler-Fox, C.S. Carter, K. Hines, J.A. O'Shaughnessy, S.F. Leitman, J.J. Greenblatt, K. Cowan, et al.
Retroviral Gene Transduction of Adult Peripheral Blood or Marrow-Derived CD34+ Cells for Six Hours Without Growth Factors or on Autologous Stroma Does Not Improve Marking Efficiency Assessed In Vivo
Blood, June 1, 1997; 89(11): 4040 - 4046.
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Proc. Natl. Acad. Sci. USAHome page
C. A. Blau, K. R. Peterson, J. G. Drachman, and D. M. Spencer
A proliferation switch for genetically modified cells
PNAS, April 1, 1997; 94(7): 3076 - 3081.
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L.B. To, D.N. Haylock, P.J. Simmons, and C.A. Juttner
The Biology and Clinical Uses of Blood Stem Cells
Blood, April 1, 1997; 89(7): 2233 - 2258.
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G. Bauer, P. Valdez, K. Kearns, I. Bahner, S. F. Wen, J. A. Zaia, and D. B. Kohn
Inhibition of Human Immunodeficiency Virus-1 (HIV-1) Replication After Transduction of Granulocyte Colony-Stimulating Factor-Mobilized CD34+ Cells From HIV-1-Infected Donors Using Retroviral Vectors Containing Anti-HIV-1 Genes
Blood, April 1, 1997; 89(7): 2259 - 2267.
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P. W. Hargrove, E. F. Vanin, G. J. Kurtzman, and A. W. Nienhuis
High-Level Globin Gene Expression Mediated by a Recombinant Adeno-Associated Virus Genome That Contains the 3' gamma  Globin Gene Regulatory Element and Integrates as Tandem Copies in Erythroid Cells
Blood, March 15, 1997; 89(6): 2167 - 2175.
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W. M. Weil, G. F. Linton, N. Whiting-Theobald, S. J. Vowells, S. P. Rafferty, F. Li, and H. L. Malech
Genetic Correction of p67phox Deficient Chronic Granulomatous Disease Using Peripheral Blood Progenitor Cells as a Target for Retrovirus Mediated Gene Transfer
Blood, March 1, 1997; 89(5): 1754 - 1761.
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BloodHome page
D. Dilloo, D. Rill, C. Entwistle, M. Boursnell, W. Zhong, W. Holden, M. Holladay, S. Inglis, and M. Brenner
A Novel Herpes Vector for the High-Efficiency Transduction of Normal and Malignant Human Hematopoietic Cells
Blood, January 1, 1997; 89(1): 119 - 127.
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BloodHome page
C. A. Blau, T. Neff, and T. Papayannopoulou
Cytokine Prestimulation as a Gene Therapy Strategy: Implications for Using the MDR1 Gene as a Dominant Selectable Marker
Blood, January 1, 1997; 89(1): 146 - 154.
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Proc. Natl. Acad. Sci. USAHome page
J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert
Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles
PNAS, December 24, 1996; 93(26): 15266 - 15271.
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ScienceHome page
C. Bordignon, L. D. Notarangelo, N. Nobili, G. Ferrari, G. Casorati, P. Panina, E. Mazzolari, D. Maggioni, C. Rossi, P. Servida, et al.
Gene Therapy in Peripheral Blood Lymphocytes and Bone Marrow for ADA- Immunodeficient Patients
Science, October 20, 1995; 270(5235): 470 - 475.
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NEJMHome page
A. Kessinger
Circulating Stem Cells -- Waxing Hematopoietic
N. Engl. J. Med., August 3, 1995; 333(5): 315 - 317.
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