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.

  • Research Article
  • Published:

B-cell-specific transgene expression using a self-inactivating retroviral vector with human CD19 promoter and viral post-transcriptional regulatory element

Gene Therapy volume 11, pages 992–1000 (2004)Cite this article

Abstract

Retroviral gene transfer resulting in transgene expression selectively restricted to specific cell lineages would be desirable for many gene therapeutic applications. Such transcriptional targeting of retroviruses can be accomplished by employing eukaryotic control elements in self-inactivating (SIN) retroviral vectors, but use of these vectors is complicated by an accompanying reduction in viral titers. To overcome this restriction and address the influence of the post-transcriptional regulatory element of the Woodchuck hepatitis virus (WPRE) on viral titers and transgene expression, we developed SIN-vectors with and without WPRE. Using the enhancer-promoter of the Spleen Focus Forming virus (SFFV) to direct eGFP expression to multiple hematopoietic lineages, we show that WPRE significantly (>10 ×) increased viral titers (>106 per ml of unconcentrated supernatant) and transgene expression in NIH3T3 cells in vitro. Gene expression in vivo was significantly lowered in lymphoid cells, but not in myeloid cells when WPRE was present. Furthermore, the use of WPRE in combination with the B-cell lineage-specific CD19 promoter significantly increased viral titers and allowed targeting of transgene expression by SIN-vectors specifically to B cells throughout their development in primary and secondary lymphoid organs.

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

Similar content being viewed by others

References

  1. Mulligan RC . The basic science of gene therapy. Science 1993; 260: 926–932.

    Article  CAS  PubMed  Google Scholar 

  2. Kay MA, Glorioso JC, Naldini L . Viral vectors for gene therapy: the art of turning infections agents into vehicles of therapeutics. Nat Med 2001; 7: 33–40.

    Article  CAS  PubMed  Google Scholar 

  3. Somia N, Verma IM . Gene therapy: trials and tribulations. Nat Rev Genet 2000; 1: 91–99.

    Article  CAS  PubMed  Google Scholar 

  4. Crystal RG . Transfer of genes to humans: early lessons and obstacles to success. Science 1995; 270: 404–410.

    Article  CAS  PubMed  Google Scholar 

  5. Cheng L et al. A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells. Gene Therapy 1997; 4: 1013–1022.

    Article  CAS  PubMed  Google Scholar 

  6. Emerman M, Temin HM . Quantitative analysis of gene suppression in integrated retrovirus vectors. Mol Cell Biol 1986; 6: 792–800.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Emerman M, Temin HM . Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 1984; 39: 449–467.

    Article  CAS  PubMed  Google Scholar 

  8. Persons DA, Nienhuis AW . Gene therapy for the hemoglobin disorders: past, present, and future. Proc Natl Acad Sci USA 2000; 97: 5022–5024.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Proudfoot NJ . Transcriptional interference and termination between duplicated alphaglobin gene constructs suggests a novel mechanism for gene regulation. Nature 1986; 322: 562–565.

    Article  CAS  PubMed  Google Scholar 

  10. Miller N, Whelan J . Progress in transcriptionally targeted and regulatable vectors for genetic therapy. Hum Gene Ther 1997; 8: 803–815.

    Article  CAS  PubMed  Google Scholar 

  11. Yee JK et al. Gene expression from transcriptionally disabled retroviral vectors. Proc Natl Acad Sci USA 1987; 84: 5197–5201.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Soriano P, Friedrich G, Lawinger P . Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J Virol 1991; 65: 2314–2319.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Yu SF et al. Self-inactivating retroviral vectors designed for transfer of whole genes into mamalian cells. Proc Natl Acad Sci USA 1986; 83: 3194–3198.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Donello JE et al. The hepatitis B virus posttranscriptional regulatory element is composed of two subelements. J Virol 1996; 70: 4345–4351.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Huang J, Liang TJ . A novel hepatitis B Virus (HBV) genetic element with Rev response element like properties that is essential for expression of HBV gene products. Mol Cell Biol 1993; 13: 7476–7486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Schwenter F, Deglon N, Aebischer P . Optimization of human erythropoietin secretion from MLV-infected human primary fibroblasts used for encapsulated cell therapy. J Gene Med 2003; 5: 246–257.

    Article  CAS  PubMed  Google Scholar 

  17. Moreau-Gaudry F et al. High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors. Blood 2001; 98: 2664–2672.

    Article  CAS  PubMed  Google Scholar 

  18. Ramezani A, Hawley TS, Hawley RG . Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol Ther 2000; 2: 458–469.

    Article  CAS  PubMed  Google Scholar 

  19. Schambach A et al. Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors. Mol Ther 2000; 2: 435–445.

    Article  CAS  PubMed  Google Scholar 

  20. Zufferey R, Donello JE, Trono D, Hope TJ . Woodchuck hepatitis virus posttrancriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 1999; 73: 2886–2892.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Huang Y, Wimler KM, Carmichael GG . Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing. EMBO J 1999; 18: 1642–1652.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Donello JE, Loeb JE, Hope TJ . Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J Virol 1998; 72: 5085–5092.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Krop I, Shaffer AL, Fearon DT, Schlissel MS . The signaling activity of murine CD19 is regulated during cell development. J Immunol 1996; 157: 48–56.

    CAS  PubMed  Google Scholar 

  24. Rickert RC, Rajewsky K, Roes J . Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 1995; 376: 352–355.

    Article  CAS  PubMed  Google Scholar 

  25. Krop I et al. Self-renewal of B-1 lymphocytes is dependent on CD19. Eur J Immunol 1996; 26: 238–242.

    Article  CAS  PubMed  Google Scholar 

  26. Sato S, Steeber DA, Jansen PJ, Tedder TF . CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogeneous CD19. J Immunol 1997; 158: 4662–4669.

    CAS  PubMed  Google Scholar 

  27. Inaoki M et al. CD19-regulated signaling thresholds control peripheral tolerance and autoantibody production in B lymphocytes. J Exp Med 1997; 186: 1923–1931.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kleindienst P, Chretien I, Winkler T, Brocker T . Functional comparison of thymic B cells and dendritic cells in vivo. Blood 2000; 95: 2610–2616.

    CAS  PubMed  Google Scholar 

  29. Baum C et al. Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells. J Virol 1995; 69: 7541–7547.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Kozmik Z et al. The promoter of the CD19 gene is a target for the B-Cell-specific transcription factor BSAP. Mol Cell Biol 1992; 12: 2662–2672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gisler R, Akerblad P, Sigvardsson M . A human early B-cell factor-like protein participates in the regulation of the human CD19 promoter. Mol Immunol 1999; 36: 1067–1077.

    Article  CAS  PubMed  Google Scholar 

  32. Cupelli L, Okenquist SA, Trubetskoy A, Lenz J . The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression. J Virol 1998; 72: 7807–7814.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Zaiss AK, Son S, Chang LJ . RNA 3′ readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy. J Virol 2002; 76: 7209–7219.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Furger A, Monks J, Proudfoot NJ . The retroviruses human immunodeficiency virus type 1 and Moloney murine leukemia virus adopt radically different strategies to regulate promoter-proximal polyadenylation. J Virol 2001; 75: 11735–11746.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zufferey R et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998; 72: 9873–9880.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Naldini L . Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol 1998; 9: 457–463.

    Article  CAS  PubMed  Google Scholar 

  37. Miyoshi H et al. Development of a self-inactivating lentivirus vector. J Virol 1998; 72: 8150–8157.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Mautino MR, Morgan RA . Enhanced inhibition of human immunodeficiency virus type 1 envelope antisense RNA. Hum Gene Ther 2002; 13: 1027–1037.

    Article  CAS  PubMed  Google Scholar 

  39. Salmon P et al. High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. Blood 2000; 96: 3392–3398.

    CAS  PubMed  Google Scholar 

  40. Gropp M et al. stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol Ther 2003; 7: 281–287.

    Article  CAS  PubMed  Google Scholar 

  41. Yam PY et al. Design of HIV vectors for efficient gene delivery into human hematopoietic cells. Mol Ther 2002; 5: 479–484.

    Article  CAS  PubMed  Google Scholar 

  42. Huang ZM, Zang WQ, Yen TS . Cellular proteins that bind to the hepatitis B virus posttranscriptional regulatory element. Virology 1996; 217: 573–581.

    Article  CAS  PubMed  Google Scholar 

  43. Popa I, Harris ME, Donello JE, Hope TJ . CRM1-dependent function of a cis-acting RNA export element. Mol Cell Biol 2002; 22: 2057–2067.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Grande A et al. Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells. Blood 1999; 93: 3276–3285.

    CAS  PubMed  Google Scholar 

  45. Cui Y et al. Trageting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells. Blood 2002; 99: 399–408.

    Article  CAS  PubMed  Google Scholar 

  46. Marodon G et al. Specific transgene expression in human and mouse CD4+ cells using lentiviral vectors with regulatory sequences from the CD4 gene. Blood 2003; 101: 3416–3423.

    Article  CAS  PubMed  Google Scholar 

  47. Lutzko C et al. Lentivirus vectors incorporating the immunoglobulin heavy chain enhacer and matrix attachment regions provide position-independent expression in B lymphocytes. J Virol 2003; 77: 7341–7351.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Challita PM, Kohn DB . Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo. Proc Natl Acad Sci USA 1994; 91: 2567–2571.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lange C, Blankenstein T . Loss of retroviral gene expression in bone marrow reconstituted mice correlates with down-regulation of gene expression in long-term culture initiating cells. Gene Therapy 1997; 4: 303–308.

    Article  CAS  PubMed  Google Scholar 

  50. Hofer M et al. Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain. EMBO J 1990; 9: 2459–2464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge Elias Hobeika for providing the B-cell line mb-1 MCM:mEGFP and Claudia Hartmann for her kind support with the quantitative real-time PCR. We thank A Bol and W Mertl for excellent care of mice and Kristen Kerksiek for critical reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ba 1837/4/SFB 571 and SFB 456).

Author information

Authors and Affiliations

  1. Institute for Immunology, Ludwig-Maximilians-University, Munich, Germany

    M Werner & T Brocker

  2. Department of Hematology and Oncology, Hannover Medical School, Hannover, Germany

    J Kraunus & C Baum

Authors
  1. M Werner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Kraunus
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C Baum
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T Brocker
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Werner, M., Kraunus, J., Baum, C. et al. B-cell-specific transgene expression using a self-inactivating retroviral vector with human CD19 promoter and viral post-transcriptional regulatory element. Gene Ther 11, 992–1000 (2004). https://doi.org/10.1038/sj.gt.3302255

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302255

Keywords

This article is cited by

Search

Advanced search

Quick links