Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome

Nicholas Hubbard, David Hagin, Karen Sommer, Yumei Song, Iram Khan, Courtnee Clough, Hans D. Ochs, David J. Rawlings, Andrew M. Scharenberg and Troy R. Torgerson

Key Points

  • The CD40LG locus can be specifically targeted and repaired in primary human T cells by insertion of a spliced CD40LG complementary DNA.

  • Gene editing restores regulated CD40L expression in X-HIGM T cells, reconstituting B-cell immunoglobulin class switching.

Publisher's Note: There is an Inside Blood Commentary on this article in this issue.


Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter–dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease–induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3′-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40–murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome.

  • Submitted November 20, 2015.
  • Accepted February 10, 2016.
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