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