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In vivo selection of retrovirally transduced hematopoietic stem cells

Nature Medicine volume 4, pages 1136–1143 (1998)Cite this article

Abstract

One of the main impediments to effective gene therapy of blood disorders is the resistance of human hematopoietic stem cells to stable genetic modification. We show here that a small minority of retrovirally transduced stem cells can be selectively enriched in vivo, which might be a way to circumvent this obstacle. We constructed two retroviral vectors containing an antifolate-resistant dihydrofolate reductase cDNA transcriptionally linked to a reporter gene. Mice were transplanted with transduced bone marrow cells and then treated with an antifolate-based regimen that kills unmodified stem cells. Drug treatment significantly increased the percentage of vector-expressing peripheral blood erythrocytes, platelets, granulocytes, and T and B lymphocytes. Secondary transplant experiments demonstrated that selection occurred at the level of hematopoietic stem cells. This system for in vivo stem-cell selection provides a means to increase the number of genetically modified cells after transplant, and may circumvent an substantial obstacle to successful gene therapy for human blood diseases.

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Figure 1: Sequential analysis of the percentage of CD24-expressing peripheral blood RBCs and platelets in transplanted mice treated with various anti-metabolites.
Figure 2: Hemoglobin phenotype analysis of mice treated with TMTX plus NBMPR-P and untreated mice.
Figure 3: Flow cytometric analysis of GFP expression in peripheral blood cells.
Figure 4: Sequential flow cytometric analysis of the percentage of GFP-expressing peripheral blood cells from multiple hematopoietic lineages.
Figure 5: GFP expression in peripheral blood cells of secondary transplant recipients.
Figure 6: Southern blot analysis of genomic DNA from secondary transplant mice.

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Acknowledgements

The authors thank E. Allay for her technical assistance, E. Wingfield and A. M. Hamilton-Easton for their assistance with the flow cytometry analyses, and A. Nienhuis for critical review of the manuscript. We also thank AMGEN (Thousand Oaks, California) for their gift of rat SCF and human IL-6. This work was supported in part by National Heart, Lung, and Blood Institute Program Project Grant No. P01 HL 53749 (BPS), The James S. McDonnell Foundation Grant No. 94-50 (BPS), The John H. Sununu Postdoctoral Fellowship (JAA), The Assisi Foundation of Memphis Grant 94-00, US Public Health Service Grant No. P01 CA 31922 (RLB), Cancer Center Support Grant No. P30 CA 21765 (BPS, RLB), and the American Lebanese Syrian Associated Charities (ALSAC)

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Authors and Affiliations

  1. Department of Hematology and Oncology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis , 38105, Tennessee

    James A. Allay, Derek A. Persons, Jacques Galipeau & Brian P. Sorrentino

  2. Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, 38105, Tennessee

    Janice M. Riberdy

  3. Department of Experimental Oncology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis , 38105, Tennessee

    Richard A. Ashmun

  4. Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis , 38105, Tennessee

    Raymond L. Blakley

  5. Department of Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, 38105, Tennessee

    Brian P. Sorrentino

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Allay, J., Persons, D., Galipeau, J. et al. In vivo selection of retrovirally transduced hematopoietic stem cells. Nat Med 4, 1136–1143 (1998). https://doi.org/10.1038/2632

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