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

Abstract

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.

Evolving Gene Therapy in Primary Immunodeficiency

“…Proof of concept has now been demonstrated in HSC/Ps from SCID-X1 and CGD patients (at safe harbor and at natural gene locus) and for XHIGM in T cells. [113][114][115] One intriguing recent report has used a gene-editing strategy to convert a mutant NCF1 pseudogene into a functional gene, thereby permitting correction of all molecular variants of p47phox-deficient CGD through a single approach. 116 Although functional correction through editing has now been demonstrated in induced pluripotent stem cells (iPSCs) and human HSC/Ps, it still probably remains limited by efficiency in true HSCs, which is required for sustained clinical effect in many PIDs.…”

Engineering HIV-Resistant, Anti-HIV Chimeric Antigen Receptor T Cells

“…11,46 Using new gene-editing techniques, it has recently become possible to achieve high rates of homology-directed recombination (HDR) of therapeutic cassettes into targeted loci, including CCR5 in primary T cells. [47][48][49][50] We have previously shown introduction of cDNA expression cassettes at the CCR5 locus in primary human T cells using an mRNA-delivered megaTAL nuclease and a homologous AAV donor template at rates of up to 60%. 48 HDR has the potential advantage of simultaneous introduction of a CAR and disruption of CCR5 to protect engineered cells from HIV.…”

Genome and Epigenome Editing to Treat Disorders of the Hematopoietic System

“…In 2016, Torgerson's group used TALENs to restore CD40L function. 17 Mutations in the CD40LG locus result in an inability to undergo immunoglobulin class switch and are associated with hyper-IgM (HIGM) syndrome. Previous attempts to perform gene therapy with retroviral delivery of a CD40L expression cassette to mouse HSCs were able to restore adaptive immunity but treated mice developed thymic lymphoproliferative disorder later on.…”