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


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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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.

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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).

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