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Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates

Abstract

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34+ cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking1,2. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification–mediated PCR (LAM-PCR) to identify clonal vector proviral integrants3,4. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID)5,6.

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Figure 1: LAM-PCR.
Figure 2: Quantification of the contribution of the predominant clone to total gene marking.
Figure 3: Analysis of T-cell clones.

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References

  1. Kohn, D.B. et al. Engraftment of gene-modified cells from umbilical cord blood in neonates with adenosine deaminase deficiency. Nat. Med. 1, 1017–1026 (1995).

    Article  CAS  Google Scholar 

  2. Kohn D.B. et al. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates. Nat. Med. 4, 775–780 (1998).

    Article  CAS  Google Scholar 

  3. Schmidt, M. et al. Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum. Gene Ther. 12, 743–749 (2001).

    Article  CAS  Google Scholar 

  4. Schmidt, M. et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 100, 2737–2743 (2002).

    Article  CAS  Google Scholar 

  5. Hacein-Bey-Abina, S. et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255–256 (2003).

    Article  Google Scholar 

  6. Marshall, E. Gene therapy. Second child in French trial is found to have leukemia. Science 299, 320 (2003).

    Article  CAS  Google Scholar 

  7. Capel, B., Hawley, R.G. & Mintz, B. Long- and short-lived murine hematopoietic stem cell clones individually identified with retroviral integration markers. Blood 75, 2267–2270 (1990).

    CAS  PubMed  Google Scholar 

  8. Morrison, S.J. & Weissman, I.L. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1, 661–673 (1994).

    Article  CAS  Google Scholar 

  9. Glimm, H. et al. Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-β2 microglobulin-null mice. J. Clin. Invest. 107, 199–206 (2001).

    Article  CAS  Google Scholar 

  10. Willenbrock, K. et al. Analysis of T-cell subpopulations in T-cell non-Hodgkin's lymphoma of angioimmunoblastic lymphadenopathy with dysproteinemia type by single target gene amplification of T cell receptor-β gene rearrangements. Am. J. Pathol. 158, 1851–1857 (2001).

    Article  CAS  Google Scholar 

  11. Tisdale, J.F. et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 92, 1131–1141 (1998).

    CAS  PubMed  Google Scholar 

  12. Kim, H.J. et al. Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates. Blood 96, 1–8 (2000).

    CAS  PubMed  Google Scholar 

  13. Jensen, M.C. et al. Human T lymphocyte genetic modification with naked DNA. Mol. Ther. 1, 49–55 (2000).

    Article  CAS  Google Scholar 

  14. Kent, W.J. BLAT: the BLAST-like Alignment Tool. Genome Res. 12, 656–664 (2002).

    Article  CAS  Google Scholar 

  15. Kent, W.J. et al. The Human Genome Browser at UCSC. Genome Res. 12, 996–1006 (2002).

    Article  CAS  Google Scholar 

  16. Lander, E. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–892 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Jensen (City of Hope National Medical Center) for assistance with T-cell cloning. This research was supported by US National Institutes of Health grants 1P50 HL54850 and 3 MO1 RR0043-35S1 and by grants Ka 976/4-1 from the Deutsche Forschungsgemeinschaft and 01KV9527/7 from the German Minister for Education and Research. D.B.K. is the recipient of a Distinguished Clinical Scientist Award from the Doris Duke Charitable Foundation.

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Correspondence to Donald B. Kohn.

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Schmidt, M., Carbonaro, D., Speckmann, C. et al. Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates. Nat Med 9, 463–468 (2003). https://doi.org/10.1038/nm844

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