Summary X-linked 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 resulted in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the TALEN and 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 (HSC) as well as in cell lines and XHIM patient-derived T cells. Notably, gene corrected HSC engrafted in immunodeficient mice at clinically-relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

No abstract

No abstract

The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human β- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in β-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the β- and δ-globin paralogs when delivering these nucleases. Pooled CD34 cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34 cells and colony-forming units, the intergenic β-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target β-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations.