Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM. [Display omitted] •TALENs and CRISPRs allow targeted gene insertion at CD40LG in hematopoietic stem cells•Adenoviral helper proteins augment rates of gene modification in vitro, but not in vivo•Gene-modified HSCs engraft in mice at clinically relevant frequencies Kuo et al. demonstrate that both TALEN and CRISPR/Cas9 platforms combined with a corrective AAV donor can mediate site-specific gene editing at the CD40 ligand locus. High levels of gene modification were achieved in human hematopoietic stem cells, which engraft in immunodeficient mice at clinically relevant frequencies.