Skip to main content

Advertisement

SpringerLink
Log in

Suppression of HLA expression by lentivirus-mediated gene transfer of siRNA cassettes and in vivo chemoselection to enhance hematopoietic stem cell transplantation

  • Published:
Immunologic Research Aims and scope Submit manuscript

Abstract

Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation. The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing. Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps “universally” compatible cellular grafts.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Sands MS, Barker JE, Vogler C, Levy B, Gwynn B, Galvin N, et al. Treatment of murine mucopolysaccharidosis type VII by syngeneic bone marrow transplantation in neonates. Lab Invest. 1993;68:676–86.

    PubMed  CAS  Google Scholar 

  2. Petersdorf EW, Malkki M. Human leukocyte antigen matching in unrelated donor hematopoietic cell transplantation. Semin Hematol. 2005;42:76–84.

    Article  PubMed  CAS  Google Scholar 

  3. Petersdorf EW, Hansen JA, Martin PJ, Woolfrey A, Malkki M, Gooley T, et al. Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation. N Engl J Med. 2001;345:1794–800.

    Article  PubMed  CAS  Google Scholar 

  4. Tiercy JM, Villard J, Roosnek E. Selection of unrelated bone marrow donors by serology, molecular typing and cellular assays. Transpl Immunol. 2002;10:215–21.

    Article  PubMed  CAS  Google Scholar 

  5. Fleischhauer K, Kernan NA, O’Reilly RJ, Dupont B, Yang SY. Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44. N Engl J Med. 1990;323:1818–22.

    Article  PubMed  CAS  Google Scholar 

  6. Petersdorf EW, Anasetti C, Martin PJ, Gooley T, Radich J, Malkki M, et al. Limits of HLA mismatching in unrelated hematopoietic cell transplantation. Blood. 2004;104:2976–80.

    Article  PubMed  CAS  Google Scholar 

  7. Sasazuki T, Juji T, Morishima Y, Kinukawa N, Kashiwabara H, Inoko H, et al. Effect of matching of class I HLA alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. Japan Marrow Donor Program. N Engl J Med. 1998;339:1177–85.

    Article  PubMed  CAS  Google Scholar 

  8. Rubinstein P. HLA matching for bone marrow transplantation—how much is enough? N Engl J Med. 2001;345:1842–4.

    Article  PubMed  CAS  Google Scholar 

  9. Vargas-Diez E, Fernandez-Herrera J, Marin A, Camara R, Garcia-Diez A. Analysis of risk factors for acute cutaneous graft-versus-host disease after allogeneic stem cell transplantation. Br J Dermatol. 2003;148:1129–34.

    Article  PubMed  CAS  Google Scholar 

  10. McDonald RA, Smith JM, Ho M, Lindblad R, Ikle D, Grimm P, et al. Incidence of PTLD in pediatric renal transplant recipients receiving basiliximab, calcineurin inhibitor, sirolimus and steroids. Am J Transplant. 2008;8:984–9.

    Article  PubMed  CAS  Google Scholar 

  11. Abed N, Casper JT, Camitta BM, Margolis D, Trost B, Orentas R, et al. Evaluation of histogenesis of B-lymphocytes in pediatric EBV-related post-transplant lymphoproliferative disorders. Bone Marrow Transplant. 2004;33:321–7.

    Article  PubMed  CAS  Google Scholar 

  12. Freland S, Chambers BJ, Andersson M, Van Kaer L, Ljunggren HG. Rejection of allogeneic and syngeneic but not MHC class I-deficient tumor grafts by MHC class I-deficient mice. J Immunol. 1998;160:572–9.

    PubMed  CAS  Google Scholar 

  13. Pollak R, Blanchard JM. Organ donor or graft pretreatment to prolong allograft survival: lessons learned in the murine model. Transplantation. 2000;69:2432–9.

    Article  PubMed  CAS  Google Scholar 

  14. Mhashilkar AM, Doebis C, Seifert M, Busch A, Zani C, Soo Hoo J, et al. Intrabody-mediated phenotypic knockout of major histocompatibility complex class I expression in human and monkey cell lines and in primary human keratinocytes. Gene Ther. 2002;9:307–19.

    Article  PubMed  CAS  Google Scholar 

  15. Beyer F, Doebis C, Busch A, Ritter T, Mhashilkar A, Marasco WM, et al. Decline of surface MHC I by adenoviral gene transfer of anti-MHC I intrabodies in human endothelial cells-new perspectives for the generation of universal donor cells for tissue transplantation. J Gene Med. 2004;6:616–23.

    Article  PubMed  CAS  Google Scholar 

  16. Drukker M, Benvenisty N. The immunogenicity of human embryonic stem-derived cells. Trends Biotechnol. 2004;22:136–41.

    Article  PubMed  CAS  Google Scholar 

  17. Bradley JA, Bolton EM, Pedersen RA. Stem cell medicine encounters the immune system. Nat Rev Immunol. 2002;2:859–71.

    Article  PubMed  CAS  Google Scholar 

  18. Hammond SM, Caudy AA, Hannon GJ. Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet. 2001;2:110–19.

    Article  PubMed  CAS  Google Scholar 

  19. McManus MT, Petersen CP, Haines BB, Chen J, Sharp PA. Gene silencing using micro-RNA designed hairpins. RNA. 2002;8:842–50.

    Article  PubMed  CAS  Google Scholar 

  20. Gonzalez S, Castanotto D, Li H, Olivares S, Jensen MC, Forman SJ, et al. Amplification of RNAi—targeting HLA mRNAs. Mol Ther. 2005;11:811–18.

    Article  PubMed  CAS  Google Scholar 

  21. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–19.

    Article  PubMed  CAS  Google Scholar 

  22. Fischer A, Abina SH, Thrasher A, von Kalle C, Cavazzana-Calvo M. LMO2 and gene therapy for severe combined immunodeficiency. N Engl J Med. 2004;350:2526–7. author reply 2526–2527.

    Article  PubMed  CAS  Google Scholar 

  23. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998;72:8463–71.

    PubMed  CAS  Google Scholar 

  24. Haga K, Lemp N, Logg CR, Nagashima J, Faure-Kumar E, Gomez GG, et al. Permanent, lowered HLA Class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes. Transpl Proc. 2006;38:3184–8.

    Article  CAS  Google Scholar 

  25. Figueiredo C, Seltsam A, Blasczyk R. Class-, gene-, and group-specific HLA silencing by lentiviral shRNA delivery. J Mol Med. 2006;84:425–37.

    Article  PubMed  CAS  Google Scholar 

  26. Figueiredo C, Horn PA, Blasczyk R, Seltsam A. Regulating MHC expression for cellular therapeutics. Transfusion. 2007;47:18–27.

    Article  PubMed  CAS  Google Scholar 

  27. Kittler EL, Peters SO, Crittenden RB, Debatis ME, Ramshaw HS, Stewart FM, et al. Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts. Blood. 1997;90:865–72.

    PubMed  CAS  Google Scholar 

  28. Traycoff CM, Cornetta K, Yoder MC, Davidson A, Srour EF. Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential. Exp Hematol. 1996;24:299–306.

    PubMed  CAS  Google Scholar 

  29. Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ. Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol. 1995;23:461–9.

    PubMed  CAS  Google Scholar 

  30. Bunting KD, Galipeau J, Topham D, Benaim E, Sorrentino BP. Transduction of murine bone marrow cells with an MDR1 vector enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferative syndrome in transplanted mice. Blood. 1998;92:2269–79.

    PubMed  CAS  Google Scholar 

  31. Cellot S, Krosl J, Chagraoui J, Meloche S, Humphries RK, Sauvageau G. Sustained in vitro trigger of self-renewal divisions in Hoxb4hiPbx1(10) hematopoietic stem cells. Exp Hematol. 2007;35:802–16.

    Article  PubMed  CAS  Google Scholar 

  32. Zhang XB, Beard BC, Trobridge GD, Wood BL, Sale GE, Sud R, et al. High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector. J Clin Invest. 2008;118:1502–10.

    Article  PubMed  CAS  Google Scholar 

  33. Davis BM, Roth JC, Liu L, Xu-Welliver M, Pegg AE, Gerson SL. Characterization of the P140 K, PVP(138–140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy. Hum Gene Ther. 1999;10:2769–78.

    Article  PubMed  CAS  Google Scholar 

  34. Sawai N, Zhou S, Vanin EF, Houghton P, Brent TP, Sorrentino BP. Protection and in vivo selection of hematopoietic stem cells using temozolomide, O6-benzylguanine, and an alkyltransferase-expressing retroviral vector. Mol Ther. 2001;3:78–87.

    Article  PubMed  CAS  Google Scholar 

  35. Persons DA, Allay ER, Sawai N, Hargrove PW, Brent TP, Hanawa H, et al. Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells. Blood. 2003;102:506–13.

    Article  PubMed  CAS  Google Scholar 

  36. Zielske SP, Reese JS, Lingas KT, Donze JR, Gerson SL. In vivo selection of MGMT(P140 K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning. J Clin Invest. 2003;112:1561–70.

    PubMed  CAS  Google Scholar 

  37. Bowman JE, Reese JS, Lingas KT, Gerson SL. Myeloablation is not required to select and maintain expression of the drug-resistance gene, mutant MGMT, in primary and secondary recipients. Mol Ther. 2003;8:42–50.

    Article  PubMed  CAS  Google Scholar 

  38. Pollok KE, Hartwell JR, Braber A, Cooper RJ, Jansen M, Ragg S, et al. In vivo selection of human hematopoietic cells in a xenograft model using combined pharmacologic and genetic manipulations. Hum Gene Ther. 2003;14:1703–14.

    Article  PubMed  CAS  Google Scholar 

  39. Neff T, Horn PA, Peterson LJ, Thomasson BM, Thompson J, Williams DA, et al. Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model. J Clin Invest. 2003;112:1581–8.

    PubMed  CAS  Google Scholar 

  40. Neff T, Beard BC, Peterson LJ, Anandakumar P, Thompson J, Kiem HP. Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy. Blood. 2005;105:997–1002.

    Article  PubMed  CAS  Google Scholar 

  41. Zielske SP, Gerson SL. Lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro. Mol Ther. 2002;5:381–7.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to acknowledge the researchers in the Kasahara and Gaensler Laboratories who have contributed to this work: In the Kasahara Laboratory, Dr. Kazunori Haga and Nathan Lemp contributed equally as the primary researchers who performed the studies described in this review; Dr. Christopher R. Logg designed the original shRNA constructs, and supervised their cloning and testing in lentiviral vectors by Jill Nagashima; Dr. Emmanuelle Faure-Kumar performed large-scale vector production; Dr. Renata Stripecke provided assistance in data analysis and helpful discussion, and the UCLA Vector Shared Resource/Core facility provided technical assistance. We would also like to thank our collaborators, Dr. James Cicciarelli and Dr. Robert Mendez at the National Institute of Transplantation (NIT) at St. Vincent’s Medical Center in Los Angeles, Dr. German G. Gomez and Dr. Carol Kruse at the Sidney Kimmel Cancer Center in San Diego, and Dr. William McBride and Dr. Richard Gatti at the UCLA Center for Biological Radioprotectors. Work in the Kasahara laboratory was supported in part by the NIAID-funded UCLA Center for Biological Radioprotectors (U19 AI 067769), the NIT Foundation, and the California Institute for Regenerative Medicine (RS1-00402-01). Researchers in the Gaensler laboratory who have contributed to this work include Dr. Linda Flebbe-Rehwaldt who developed the initial neonatal transplantation model including the non-ablative preparative regimen and in vivo chemoselection protocols; Dr. Rustom Falahati performed the in vitro chemoselection assay, optimized conditions for lentiviral transduction and transplantation of transduced HSC, and performed FACS and immunological analyses. We would like to thank our collaborators Dr. Stanton Gerson at the Department of Medicine, Division of Hematology–Oncology, Case Western Reserve University, Cleveland, Ohio, who provided the MAG vector, and Dr. William Murphy at the Department of Microbiology and Immunology, University of Nevada, Reno, Nevada for valuable advice. The work in the Gaensler laboratory was supported by NIH 5R21 DK069430-02, NIH 1R01HL082665-01A2, and a UCSF Bridge Funding award.

Author information

Authors and Affiliations

  1. Department of Medicine, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA, USA

    Katrin Hacke & Noriyuki Kasahara

  2. Department of Medicine, Division of Hematology/Oncology, University of California San Francisco (UCSF), 533 Parnassus Ave., M-1286, San Francisco, CA, 94143, USA

    Rustom Falahati, Linda Flebbe-Rehwaldt & Karin M. L. Gaensler

Authors
  1. Katrin Hacke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rustom Falahati
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Linda Flebbe-Rehwaldt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Noriyuki Kasahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karin M. L. Gaensler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karin M. L. Gaensler.

Additional information

R. Falahati and K. Hacke contributed equally to this article.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hacke, K., Falahati, R., Flebbe-Rehwaldt, L. et al. Suppression of HLA expression by lentivirus-mediated gene transfer of siRNA cassettes and in vivo chemoselection to enhance hematopoietic stem cell transplantation. Immunol Res 44, 112–126 (2009). https://doi.org/10.1007/s12026-008-8088-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12026-008-8088-z

Keywords

Search

Navigation