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.

  • Article
  • Published:

Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency

Nature Medicine volume 10, pages 835–841 (2004)Cite this article

Abstract

X-linked immunodeficiency with hyper-IgM (HIGM1), characterized by failure of immunoglobulin isotype switching, is caused by mutations of the CD40 ligand (CD40L), which is normally expressed on activated CD4+ T cells. As constitutive expression of CD40L induces lymphomas, we corrected the mutation while preserving the natural regulation of CD40L using pre-mRNA trans-splicing. Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer encoding the normal CD40L exons 2–5 and was administered to syngenic CD40L-knockout mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG1 responses to keyhole limpet hemocyanin immunization, regulated CD4+ T-cell CD40L expression after CD3 stimulation in primary and secondary transplanted mice, attenuation of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative disease over 1 year. Thus, HIGM1 can be corrected by CD40L trans-splicing, leading to functional correction of the genetic defect without the adverse consequences of unregulated expression of the CD40L gene.

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: Schematic illustration of the strategy used for trans-splicing of CD40L mRNA in vivo.
Figure 2: Ex vivo trans-splicing-mediated correction of CD40L pre-mRNA in CD40L-knockout mice.
Figure 3: Partial restoration of immunoglobulin subclass switching mediated by trans-spliced CD40L in response to a thymus-dependent antigen (KLH) in vivo.
Figure 4: Analysis of CD40L expression in CD4+ T cells from CD40L trans-spliced mice.
Figure 5: Trans-spliced, CD40L-mediated attenuation of P. carinii infection in CD40L-knockout mice.

Similar content being viewed by others

References

  1. Callard, R.E., Armitage, R.J., Fanslow, W.C. & Spriggs, M.K. CD40 ligand and its role in X-linked hyper-IgM syndrome. Immunol. Today 14, 559–564 (1993).

    Article  CAS  PubMed  Google Scholar 

  2. Kroczek, R.A. et al. Defective expression of CD40 ligand on T cells causes “X-linked immunodeficiency with hyper-IgM (HIGM1)”. Immunol. Rev. 138, 39–59 (1994).

    Article  CAS  PubMed  Google Scholar 

  3. Di Santo, J.P., Bonnefoy, J.Y., Gauchat, J.F., Fischer, A. & de Saint, B.G. CD40 ligand mutations in X-linked immunodeficiency with hyper-IgM. Nature 361, 541–543 (1993).

    Article  CAS  Google Scholar 

  4. Korthauer, U. et al. Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature 361, 539–541 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Roy, M., Waldschmidt, T., Aruffo, A., Ledbetter, J.A. & Noelle, R.J. The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells. J. Immunol. 151, 2497–2510 (1993).

    CAS  PubMed  Google Scholar 

  6. Stout, R.D., Suttles, J., Xu, J., Grewal, I.S. & Flavell, R.A. Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice. J. Immunol. 156, 8–11 (1996).

    CAS  PubMed  Google Scholar 

  7. Grewal, I.S. & Flavell, R.A. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16, 111–135 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Straw, A.D., MacDonald, A.S., Denkers, E.Y. & Pearce, E.J. CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses. J. Immunol. 170, 727–734 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Gennery, A.R. et al. Treatment of CD40 ligand deficiency by hematopoietic stem cell transplantation: a survey of the European experience, 1993-2002. Blood 103, 1152–1157 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Levy, J. et al. Clinical spectrum of X-linked hyper-IgM syndrome. J. Pediatr. 131, 47–54 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Hadzic, N. et al. Correction of the hyper-IgM syndrome after liver and bone marrow transplantation. N. Engl. J. Med. 342, 320–324 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Thomas, C. et al. Brief report: correction of X-linked hyper-IgM syndrome by allogeneic bone marrow transplantation. N. Engl. J. Med. 333, 426–429 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Brown, M.P. et al. Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice. Nat. Med. 4, 1253–1260 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Sacco, M.G. et al. Lymphoid abnormalities in CD40 ligand transgenic mice suggest the need for tight regulation in gene therapy approaches to hyper immunoglobulin M (IgM) syndrome. Cancer Gene Ther. 7, 1299–1306 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Sullenger, B.A. Targeted genetic repair: an emerging approach to genetic therapy. J. Clin. Invest. 112, 310–311 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Puttaraju, M., Jamison, S.F., Mansfield, S.G., Garcia-Blanco, M.A. & Mitchell, L.G. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat. Biotechnol. 17, 246–252 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Garcia-Blanco, M.A., Puttaraju, M., Mansfield, S.G. & Mitchell, L.G. Spliceosome-mediated RNA trans-splicing in gene therapy and genomics. Gene Ther. Regul. 1, 141–163 (2000).

    Article  CAS  Google Scholar 

  18. Liu, X. et al. Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Nat. Biotechnol. 20, 47–52 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Mansfield, S.G. et al. Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Ther. 7, 1885–1895 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Puttaraju, M., Di Pasquale, J., Baker, C.C., Mitchell, L.G. & Garcia-Blanco, M.A. Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing. Mol. Ther. 4, 105–114 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Chao, H. et al. Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing. Nat. Med. 9, 1015–1019 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Garcia-Blanco, M.A. Messenger RNA reprogramming by spliceosome-mediated RNA trans-splicing. J. Clin. Invest. 112, 474–480 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bonen, L. Trans-splicing of pre-mRNA in plants, animals, and protists. FASEB J. 7, 40–46 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Caudevilla, C. et al. Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver. Proc. Natl. Acad. Sci. USA 95, 12185–12190 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Xu, J. et al. Mice deficient for the CD40 ligand. Immunity. 1, 423–431 (1994).

    Article  CAS  PubMed  Google Scholar 

  26. Testi, R., D'Ambrosio, D., De Maria, R. & Santoni, A. The CD69 receptor: a multipurpose cell-surface trigger for hematopoietic cells. Immunol. Today 15, 479–483 (1994).

    Article  CAS  PubMed  Google Scholar 

  27. Kolls, J.K., Beck, J.M., Nelson, S., Summer, W.R. & Shellito, J. Alveolar macrophage release of tumor necrosis factor during murine Pneumocystis carinii pneumonia. Am. J. Respir. Cell Mol. Biol. 8, 370–376 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. Kolls, J.K. et al. IFN-γ and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells. J. Immunol. 162, 2890–2894 (1999).

    CAS  PubMed  Google Scholar 

  29. Logan, A.C., Lutzko, C. & Kohn, D.B. Advances in lentiviral vector design for gene-modification of hematopoietic stem cells. Curr. Opin. Biotechnol. 13, 429–436 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Baumgartner, R. et al. Evidence for the requirement of T cell costimulation in the pathogenesis of natural Pneumocystis carinii pulmonary infection. Microb. Pathog. 33, 193–201 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Campbell, K.A. et al. CD40 ligand is required for protective cell-mediated immunity to Leishmania major. Immunity 4, 283–289 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. MacDonald, A.S. et al. Impaired Th2 development and increased mortality during Schistosoma mansoni infection in the absence of CD40/CD154 interaction. J. Immunol. 168, 4643–4649 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Netea, M.G., Meer, J.W., Verschueren, I. & Kullberg, B.J. CD40/CD40 ligand interactions in the host defense against disseminated Candida albicans infection: the role of macrophage-derived nitric oxide. Eur. J. Immunol. 32, 1455–1463 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Soong, L. et al. Disruption of CD40-CD40 ligand interactions results in an enhanced susceptibility to Leishmania amazonensis infection. Immunity 4, 263–273 (1996).

    Article  CAS  PubMed  Google Scholar 

  35. Stephens, J., Cosyns, M., Jones, M. & Hayward, A. Liver and bile duct pathology following Cryptosporidium parvum infection of immunodeficient mice. Hepatology 30, 27–35 (1999).

    Article  CAS  PubMed  Google Scholar 

  36. Thomsen, A.R., Nansen, A., Christensen, J.P., Andreasen, S.O. & Marker, O. CD40 ligand is pivotal to efficient control of virus replication in mice infected with lymphocytic choriomeningitis virus. J. Immunol. 161, 4583–4590 (1998).

    CAS  PubMed  Google Scholar 

  37. Merali, S., Chin, K., Grady, R.W., Weissberger, L. & Clarkson, A.B. Response of rat model of Pneumocystis carinii pneumonia to continuous infusion of deferoxamine. Antimicrob. Agents Chemother. 39, 1442–1444 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zheng, M. et al. CD4+ T cell-independent vaccination against Pneumocystis carinii in mice. J. Clin. Invest. 108, 1469–1474 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Pergolizzi, R.G. et al. In vivo trans-splicing of 5′ and 3′ segments of pre-mRNA directed by corresponding DNA sequences delivered by gene transfer. Mol. Ther. 8, 999–1008 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Armitage, R.J. et al. Molecular and biological characterization of a murine ligand for CD40. Nature 357, 80–82 (1992).

    Article  CAS  PubMed  Google Scholar 

  41. Field, J. et al. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 8, 2159–2165 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Xu, K., Ma, H., McCown, T.J., Verma, I.M. & Kafri, T. Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors. Mol. Ther. 3, 97–104 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Szilvassy, S.J., Lansdorp, P.M., Humphries, R.K., Eaves, A.C. & Eaves, C.J. Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability. Blood 74, 930–939 (1989).

    CAS  PubMed  Google Scholar 

  46. Vigna, E. et al. Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. Mol. Ther. 5, 252–261 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25, 217–222 (2000).

    Article  CAS  PubMed  Google Scholar 

  48. Kikuchi, T., Worgall, S., Singh, R., Moore, M.A. & Crystal, R.G. Dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can initiate antigen-specific humoral immunity independent of CD4+ T cells. Nat. Med. 6, 1154–1159 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank K. Nakayama and Q. Jiang for help in construction of trans-splicing vectors; A. Busch for flow cytometry analysis; N. Hackett and A. Cieciuch for real-time RT-PCR analysis; K. Kasuya for histological analysis; T. Kafri for providing plasmid; and N. Mohamed for help preparing this manuscript. These studies were supported, in part, by U01 HL66952, the Will Rogers Memorial Fund and The Malcolm Hewitt Wiener Foundation.

Author information

Authors and Affiliations

  1. Department of Genetic Medicine, Weill Medical College of Cornell University, New York, New York, USA

    Minoru Tahara, Robert G Pergolizzi, Hiroyasu Kobayashi & Ronald G Crystal

  2. Belfer Gene Therapy Core Facility, Weill Medical College of Cornell University, New York, New York, USA

    Anja Krause, Karsta Luettich & Ronald G Crystal

  3. Biostatistics Unit, North Shore–Long Island Jewish Research Institute, Manhasset, New York, USA

    Martin L Lesser

Authors
  1. Minoru Tahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert G Pergolizzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroyasu Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anja Krause
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karsta Luettich
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martin L Lesser
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ronald G Crystal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ronald G Crystal.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Schematic illustration of trans-splicer plasmid and demonstration of correction of mutated CD40L by trans-splicing in vitro. (PDF 162 kb)

Supplementary Fig. 2

Identification of the most efficient hybridization domain as assessed by in vitro trans-splicing-mediated correction of murine CD40L pre-mRNA. (PDF 53 kb)

Supplementary Fig. 3

Assessment of restoration of function following in vitro trans-splicing-mediated correction of CD40L pre-mRNA. (PDF 111 kb)

Supplementary Methods (PDF 61 kb)

Supplementary Note (PDF 46 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tahara, M., Pergolizzi, R., Kobayashi, H. et al. Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency. Nat Med 10, 835–841 (2004). https://doi.org/10.1038/nm1086

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1086

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing