Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model

Gene Therapy volume 21, pages 148–157 (2014)Cite this article

Subjects

Abstract

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) targeted gene therapy; however, RIC may be insufficient for efficient engraftment and inducing immunological tolerance to transgenes. We previously established long-term gene marking in our rhesus macaque autologous HSC transplantation model following 10 Gy total body irradiation (TBI). In this study, we evaluated RIC transplantation with 4 Gy TBI in two rhesus macaques that received equal parts of CD34+ cells transduced with green fluorescent protein (GFP)-expressing lentiviral vector and empty vector not expressing transgenes. In both animals, equivalently low gene marking between GFP and empty vectors was observed 6 months post-transplantation, even with efficient transduction of CD34+ cells in vitro. Autologous lymphocyte infusion with GFP marking resulted in an increase of gene marking in lymphocytes in a control animal with GFP tolerance, but not in the two RIC-transplanted animals. In vitro assays revealed strong cellular and humoral immune responses to GFP protein in the two RIC-transplanted animals, but this was not observed in controls. In summary, 4 Gy TBI is insufficient to permit engraftment of genetically modified HSCs and induce immunological tolerance to transgenes. Our findings should help in the design of conditioning regimens in gene therapy trials.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Aiuti A, Cattaneo F, Galimberti S, Benninghoff U, Cassani B, Callegaro L et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N Engl J Med 2009; 360: 447–458.

    Article  CAS  Google Scholar 

  2. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296: 2410–2413.

    Article  CAS  Google Scholar 

  3. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 2010; 467: 318–322.

    Article  CAS  Google Scholar 

  4. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348: 255–256.

    Article  Google Scholar 

  5. Schwarzwaelder K, Howe SJ, Schmidt M, Brugman MH, Deichmann A, Glimm H et al. Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo. J Clin Invest 2007; 117: 2241–2249.

    Article  CAS  Google Scholar 

  6. Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006; 12: 401–409.

    Article  CAS  Google Scholar 

  7. Copelan EA . Hematopoietic stem-cell transplantation. N Engl J Med 2006; 354: 1813–1826.

    Article  CAS  Google Scholar 

  8. Hsieh MM, Kang EM, Fitzhugh CD, Link MB, Bolan CD, Kurlander R et al. Allogeneic hematopoietic stem-cell transplantation for sickle cell disease. N Engl J Med 2009; 361: 2309–2317.

    Article  CAS  Google Scholar 

  9. Storb RF, Champlin R, Riddell SR, Murata M, Bryant S, Warren EH . Non-myeloablative transplants for malignant disease. Hematology Am Soc Hematol Educ Program 2001: 375–391.

  10. Antin JH . Reduced-intensity stem cell transplantation: ‘...whereof a little More than a little is by much too much.’ King Henry IV, part 1, I, 2. Hematology Am Soc Hematol Educ Program 2007: 47–54.

  11. Uchida N, Bonifacino A, Krouse AE, Metzger ME, Csako G, Lee-Stroka A et al. Accelerated lymphocyte reconstitution and long-term recovery after transplantation of lentiviral-transduced rhesus CD34(+) cells mobilized by G-CSF and plerixafor. Exp Hematol 2011; 39: 795–805.

    Article  CAS  Google Scholar 

  12. Uchida N, Hargrove PW, Lap CJ, Evans ME, Phang O, Bonifacino AC et al. High-efficiency transduction of rhesus hematopoietic repopulating cells by a modified HIV1-based lentiviral vector. Mol Ther 2012; 20: 1882–1892.

    Article  CAS  Google Scholar 

  13. Uchida N, Washington KN, Hayakawa J, Hsieh MM, Bonifacino AC, Krouse AE et al. Development of a human immunodeficiency virus type 1-based lentiviral vector that allows efficient transduction of both human and rhesus blood cells. J Virol 2009; 83: 9854–9862.

    Article  CAS  Google Scholar 

  14. Heim DA, Hanazono Y, Giri N, Wu T, Childs R, Sellers SE et al. Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model. Mol Ther 2000; 1: 533–544.

    Article  CAS  Google Scholar 

  15. Kung SK, An DS, Bonifacino A, Metzger ME, Ringpis GE, Mao SH et al. Induction of transgene-specific immunological tolerance in myeloablated nonhuman primates using lentivirally transduced CD34+ progenitor cells. Mol Ther 2003; 8: 981–991.

    Article  CAS  Google Scholar 

  16. Nienhuis AW . Development of gene therapy for blood disorders. Blood 2008; 111: 4431–4444.

    Article  CAS  Google Scholar 

  17. Persons DA . Hematopoietic stem cell gene transfer for the treatment of hemoglobin disorders. Hematology Am Soc Hematol Educ Program 2009; 1: 690–697.

    Article  Google Scholar 

  18. Bunn HF . Pathogenesis and treatment of sickle cell disease. N Engl J Med 1997; 337: 762–769.

    Article  CAS  Google Scholar 

  19. Otieno S, Bloor A, Karol R, Reichlin M, Noble RW . Specific antibodies to hemoglobin A1 (anti-Glu) and hemoglobin S (anti-Val) in the guinea pig: immunologic and structural correlations. J Immunol 1978; 121: 2458–2462.

    CAS  PubMed  Google Scholar 

  20. Uchida N, Hsieh MM, Hayakawa J, Madison C, Washington KN, Tisdale JF . Optimal conditions for lentiviral transduction of engrafting human CD34(+) cells. Gene Therapy 2011; 18: 1078–1086.

    Article  CAS  Google Scholar 

  21. Uchida N, Washington KN, Lap CJ, Hsieh MM, Tisdale JF . Chicken HS4 insulators have minimal barrier function among progeny of human hematopoietic cells transduced with an HIV1-based lentiviral vector. Mol Ther 2010; 19: 133–139.

    Article  Google Scholar 

  22. Hanawa H, Persons DA, Nienhuis AW . Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors. J Virol 2005; 79: 8410–8421.

    Article  CAS  Google Scholar 

  23. Uchida N, Hanawa H, Yamamoto M, Shimada T . The chicken HS4 core insulator blocks promoter interference in lentiviral vectors. Hum Gene Ther Methods 2013; 24: 117–124.

    Article  CAS  Google Scholar 

  24. Hanawa H, Hematti P, Keyvanfar K, Metzger ME, Krouse A, Donahue RE et al. Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system. Blood 2004; 103: 4062–4069.

    Article  CAS  Google Scholar 

  25. Uchida N, Hanawa H, Dan K, Inokuchi K, Shimada T . Leukemogenesis of b2a2-type p210 BCR/ABL in a bone marrow transplantation mouse model using a lentiviral vector. J Nippon Med Sch 2009; 76: 134–147.

    Article  CAS  Google Scholar 

  26. Tisdale JF, Hanazono Y, Sellers SE, Agricola BA, Metzger ME, Donahue RE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998; 92: 1131–1141.

    CAS  Google Scholar 

  27. Uchida N, Hsieh MM, Washington KN, Tisdale JF . Efficient transduction of human hematopoietic repopulating cells with a chimeric HIV1-based vector including SIV capsid. Exp Hematol 2013; 41: 779–788.

    Article  CAS  Google Scholar 

  28. Hanazono Y, Brown KE, Handa A, Metzger ME, Heim D, Kurtzman GJ et al. In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors. Blood 1999; 94: 2263–2270.

    CAS  PubMed  Google Scholar 

  29. Parry RV, Rumbley CA, Vandenberghe LH, June CH, Riley JL . CD28 and inducible costimulatory protein Src homology 2 binding domains show distinct regulation of phosphatidylinositol 3-kinase, Bcl-xL, and IL-2 expression in primary human CD4 T lymphocytes. J Immunol 2003; 171: 166–174.

    Article  CAS  Google Scholar 

  30. Pollok KE, Hanenberg H, Noblitt TW, Schroeder WL, Kato I, Emanuel D et al. High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments. J Virol 1998; 72: 4882–4892.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhou P, Lee J, Moore P, Brasky KM . High-efficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degrees C centrifugation and CH-296-coated plates: effect of gene transfer protocol on T cell homing receptor expression. Hum Gene Ther 2001; 12: 1843–1855.

    Article  CAS  Google Scholar 

  32. Deeks SG, Wagner B, Anton PA, Mitsuyasu RT, Scadden DT, Huang C et al. A phase II randomized study of HIV-specific T-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Mol Ther 2002; 5: 788–797.

    Article  CAS  Google Scholar 

  33. Varela-Rohena A, Molloy PE, Dunn SM, Li Y, Suhoski MM, Carroll RG et al. Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor. Nat Med 2008; 14: 1390–1395.

    Article  CAS  Google Scholar 

  34. Geraerts M, Willems S, Baekelandt V, Debyser Z, Gijsbers R . Comparison of lentiviral vector titration methods. BMC Biotechnol 2006; 6: 34.

    Article  Google Scholar 

  35. Houter JVKD. Antibody response induced after intraocular viral gene addition therapy using adeno-associated virus, lentivirus, and adenovirus vectors with the GFP Transgene in dogs. ProQuest LLC Ann Arbor, MI, USA, 2009.

Download references

Acknowledgements

This work was supported by the intramural research program of the National Heart, Lung, and Blood Institute (NHLBI) and the National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH). We thank the animal care staff and technicians at 5 Research Court for their excellent care and handling of the animals. We would also like to thank William DeGraff and Dr Jim Mitchell of the Radiation Biology Branch, National Cancer Institute (NCI) for the use of the Eldorado Cobalt-60 irradiator.

Author information

Authors and Affiliations

  1. Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD, USA

    N Uchida, R P Weitzel, M E Evans, R Green, M M Hsieh & J F Tisdale

  2. Hematology Branch, NHLBI, NIH, 5 Research Court, Rockville, MD, USA

    A C Bonifacino, A E Krouse, M E Metzger & R E Donahue

Authors
  1. N Uchida
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R P Weitzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M E Evans
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A C Bonifacino
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A E Krouse
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M E Metzger
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M M Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. R E Donahue
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J F Tisdale
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J F Tisdale.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Uchida, N., Weitzel, R., Evans, M. et al. Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model. Gene Ther 21, 148–157 (2014). https://doi.org/10.1038/gt.2013.67

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2013.67

Keywords

This article is cited by

Search

Advanced search

Quick links