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

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.

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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.

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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

Correspondence to Ronald G Crystal.

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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)

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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

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