Skip to main content
SpringerLink
Log in

Recombinant retrovirus vectors for the expression of MHC class II heterodimers

  • Original Articles
  • Published:
Surgery Today Aims and scope Submit manuscript

Abstract

Class II antigens are critical in determining the fate of vascularized allografts across major histocompatibility differences. We have recently developed a new approach to induce transplantation tolerance in miniature swine by creating MHC class II antigen “molecular chimerism” in bone marrow cells of potential recipients through retrovirus-mediated gene transfer. As part of this project, the ability of a recombinant double-expression vector (ZQ32N) to express MHC class II DQA and DQB was investigated. Flow cytometry analyses of ZQ32N transfected virus-producer cells demonstrated the cell surface expression of DQa/DQb heterodimers, thus suggesting a correct transcription, translation, and transport of the swine polypeptides to the cell surface. The analyses of RNA isolated from virus particles produced from ZQ32N transfected virus-producer cells indicated the DQ sequences to be correctly packaged. However, the DQ-negative cells transduced with the ZQ32N retrovirus did not show any DQ-retrovirus surface expression. Southern and Northern blot analyses of ZQ32N transfected and transduced cells strongly suggested DNA rearrangements and deletions which could account for transgene expression loss. An analysis of transduced cell genomes suggested DNA recombinations targeted to homologous sequences within the recombinant provirus. The implications of the sequence instability in designing vectors for gene therapy of organ transplantation are discussed.

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

Similar content being viewed by others

References

  1. Guzetta PC, Sundt TM, Suzuki T, Mixon A, Rosengard BR, Sachs DH (1991) Induction of kidney transplantation tolerance across MHC barriers by bone marrow transplantation in miniature swine. Transplantation 51:862–866

    Article  Google Scholar 

  2. Ildstad ST, Sachs DH (1984) Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 307: 168–170

    Article  CAS  PubMed  Google Scholar 

  3. Opelz G, Mytilineos J, Scherer S, Dunckley H, Trejaut J, Chapman J, Middleton D, Savage D, Fisher O, Bignon J-D, Bensa J-C, Albert E, Noreen H (1991) Survival of DNA HLA-DR typed and matched cadaver kidney transplants Lancet 338:461–463

    Article  CAS  PubMed  Google Scholar 

  4. Cho YW, Cecka JM, Terasaki, PI (1994) HLA matching effect: better survival rates and graft quality. Clin Transpl 1994:435–449

    Google Scholar 

  5. Gianello P, Fishbein JM, Sachs DH (1993) Tolerance to primary vascularized allografts in miniature swine. Immunol Rev 133:19–44

    Article  CAS  PubMed  Google Scholar 

  6. Pennington LR, Lunney JK, Sachs DH (1981) Transplantation in miniature swine: VIII. Recombination within the major histo-compatibility complex of miniature swine. Transplantation 31:66–71

    Article  CAS  PubMed  Google Scholar 

  7. Sachs DH (1992) Homozygous miniature swine. In: Swindle MM, Moody DC, Phillips LD (eds) Swine as models in biomedical research. Iowa State University Press, Ames, pp 3–15

    Google Scholar 

  8. LeGuern C, Shimada H, Emery DW, Germana S, Shafer GE, Sachs DH (1995) Retrovirus-mediated transfer of MHC class II cDNA into swine bone marrow cells. J Mol Med 73:269–278

    Article  CAS  PubMed  Google Scholar 

  9. LeGuern C, Shimada H, Emery DW, Germana S, Sachs DH (1993) Molecular transplantation of MHC genes as a means for inducing transplantation tolerance in miniature swine. Bull Inst Pasteur 91:125–133

    CAS  Google Scholar 

  10. Emery DW, SablinskiT, Shimada H, Germana S, Gianello P, Foley A, Shulman S, Arn S, Fishman J, Lorf T, Nickeleit V, Colvin RB, Sachs DH, LeGuern C (1997) Expression of an allogeneic MHC DRB trangene, through retroviral transduction of bone marrow, induces specific reduction of alloreactivity. Transplantation 64:1414–1423

    Article  CAS  PubMed  Google Scholar 

  11. Mulligan RC (1993) The basic science of gene therapy. Science 260:926–932

    Article  CAS  PubMed  Google Scholar 

  12. Miller AD (1992) Retroviral vectors. Curr Top Microbiol Immunol 158:1–24

    Article  CAS  PubMed  Google Scholar 

  13. Emery DW, Shafter GE, Karson EM, Sachs DH, LeGuern C (1993) Retrovirus-mediated transfer and expression of an allogeneic major histocompatibility complex class II DRB cDNA in swine bone marrow cultures. Blood 81:2460–2465

    CAS  PubMed  Google Scholar 

  14. Shafer GE, Emery DW, Gustafsson K, Germana S, Anderson WF, Sachs DH, LeGuern C (1991) Expression of a swine class II gene in murine bone marrow hematopoietic cells by retroviral-mediated gene transfer. Proc Natl Acad Sci USA 88:9760–9764

    Article  CAS  PubMed  Google Scholar 

  15. Fraser CC, Sykes M, Stanton-Lee R, Sachs DH, LeGuern C (1995) Specific unresponsiveness to retrovirally-transferred class I antigen is controlled through the helper pathway. J Immunol 154:1587–1595

    CAS  PubMed  Google Scholar 

  16. Sykes M, Sachs DH, Nienhuis AW, Pearson DA, Moulton AD, Bodine DM (1993) Specific prolongation of skin graft survival following retroviral transduction of bone marrow with an allogeneic major histocompatibility complex gene. Transplantation 55:197–202

    Article  CAS  PubMed  Google Scholar 

  17. McLachlin JR, Cornetta K, Eglitis MA, Anderson WF (1990) Retroviral-mediated gene transfer. Prog Nucleic Acid Res Mol Biol 38:91–135

    Article  CAS  PubMed  Google Scholar 

  18. Korman AJ, Frantz JD, Strominger JL, Mulligan RC (1987) Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc Natl Acad Sci USA 84:2150–2154

    Article  CAS  PubMed  Google Scholar 

  19. Overell RW, Weisser KE, Cosman D (1988) Stably transmitted triple-promoter retroviral vectors and their used in transformation of primary mammalian cells. Mol Cell Biol 8:1804–1808

    Google Scholar 

  20. Morgan RA, Couture L, Elroy-Stein O Ragheb J, Moss B, Anderson WF (1992) Retroviral vectors containing putative internal ribosome entry sites; development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic Acids Res 20:1293–1299

    Article  CAS  PubMed  Google Scholar 

  21. Adam MA, Ramesh N, Miller AD, Osborne WR (1991) Internal initiation of translation in retroviral vectors carrying picornavirus 5′ nontranslated regions. J Virol 65:4985–4990

    CAS  PubMed  Google Scholar 

  22. Koo HM, Brown AM, Kaufman RJ, Prorock CM, Ron Y, Dougherty JP (1992) A spleen necrosis virus-based retroviral vector which expresses two genes from a dicistronic mRNA. Virology 186:669–675

    Article  CAS  PubMed  Google Scholar 

  23. Banerjee PT, Kanor GC, Muthukumar S, Denaro M, Shimada H, Zhu S, Rosa MD, Sachs DH, LeGuern C (1997) A polycistronic retrovirus vector for expression of swine MHC class II DR alpha/beta heterodimers. Xenotransplantation 4:161–173

    Article  Google Scholar 

  24. Hantzopoulos PA, Sullenger BA, Ungers G, Gilboa E (1989) Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of the retroviral vector. Proc Natl Acad Sci USA 86:3519–3523

    Article  CAS  PubMed  Google Scholar 

  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  26. Hirsch F, Sachs DH, Gustafsson K, Pratt K, Germana S, LeGuern C (1990) Class II of the miniature swine. III. Characterization of an expressed pig class II gene homologous to HLA-DQA. Immunogenetics 31:52–56

    Article  CAS  PubMed  Google Scholar 

  27. Gustafsson K, LeGuern C, Hirsch F, Germana S, Pratt K, Sachs D (1990) Class II genes of the miniature swine: IV. Characterization and expression of two allelic class II DQB cDNA clones. J Immunol 145:1946–1951

    CAS  PubMed  Google Scholar 

  28. McKnight SL (1980) The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res 8:5949–5964

    Article  CAS  PubMed  Google Scholar 

  29. Luckow B, Schutz G (1987) CAT constructions with multiple unique restriction site for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res 15:5490–5497

    Article  CAS  PubMed  Google Scholar 

  30. Markowitz D, Goff S, Bank A (1988) A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 62:1120–1124

    CAS  PubMed  Google Scholar 

  31. Markovitz D, Goff S, Bank A (1988) Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167:400–406

    Google Scholar 

  32. Wigler M, Pellicer A, Silverstein S, Axel R (1978) Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14:725–731

    Article  CAS  PubMed  Google Scholar 

  33. Davis WC, Perryman LE, McGuire TC (1984) The identification and analysis of major functional populations of differentiated cells. In: Stern RJ, Gamble HR (eds) Hybridoma technology in agriculture and veterinary research. Rowman & Allanheld, Totowa, NJ, pp 121–144

    Google Scholar 

  34. Pescovitz MD, Lenney JK, Sachs DH (1984) Preparation and characterization of monoclonal antibodies reactive with porcine PBL. J Immunol 133:368–375

    CAS  PubMed  Google Scholar 

  35. Hirsch F, Germana S, Gustafsson K, Pratt K, Sachs DH, LeGuern C (1992) Structure and expression of class II genes in miniature swine. J Immunol 149:841–846

    CAS  PubMed  Google Scholar 

  36. Gustafsson K, Germana S, Hirsch F, Pratt K, LeGuern C, Sachs DH (1990) Structure of miniature swine class II DRB genes: conservation of hypervariable amino acid residues between distantly related mammalian species. Proc Natl Acad Sci USA 87:9798–9802

    Article  CAS  PubMed  Google Scholar 

  37. Connor J, Bannerji R, Saito S, Heston W, Fair W, Gilboa E (1993) Regression of bladder tumors in mice treated with interleukin 2 gene-modified cells. J Exp Med 177:1127–1136

    Article  CAS  PubMed  Google Scholar 

  38. Pathack V, Temin HM (1990) Broad spectrum of in vivo forward mutation, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frame-shifts, and hypermutations. Proc Natl Acad Sci USA 87:6019–6023

    Article  Google Scholar 

  39. Zhang J, Temin H (1994) Retrovirus recombination depends on the length of sequence identity and is not error prone. J Virol 68:2409–2416

    CAS  PubMed  Google Scholar 

  40. Punsinelli GA, Temin HM (1991) Characterization of large deletion occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer. J Virol 65:4786–4792

    Google Scholar 

  41. Goodrich D, Duesberg P (1990) Retroviral recombination during reverse transcription Proc Natl Acad Sci USA 87:2052–2957

    Article  CAS  PubMed  Google Scholar 

  42. Stuhlmann H, Berg P (1992) Homologous recombination of copackaged retrovirus RNAs during reverse transcription. J Virol 66:2378–2387

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, MGH-East, Building 149-9019, 13th Street, 02129, Boston, MA, USA

    Hideaki Shimada, Sharon Germana, David H. Sachs & Christian LeGuern

  2. Neuropsychiatry Branch, NIMH, Neuroscience Research Center, 2700 Martin Luther King Jr. Avenue, SE, DC 20032, Washington, USA

    Robert C. Alexander

Authors
  1. Hideaki Shimada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert C. Alexander
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sharon Germana
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David H. Sachs
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christian LeGuern
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This work was supported by NIH Grants AI33053 and HL48049

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shimada, H., Alexander, R.C., Germana, S. et al. Recombinant retrovirus vectors for the expression of MHC class II heterodimers. Surg Today 29, 533–541 (1999). https://doi.org/10.1007/BF02482348

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02482348

Key Words

Search

Navigation