Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.

Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkably high specificity, paving the way for the establishment of a potential new class of therapeutics.

Correction of IVS I-110(G>A) β-thalassemia by CRISPR/Cas-and TALEN-mediated disruption of aberrant regulatory elements in human hematopoietic stem and progenitor cells β-Hemoglobinopathies result from mutations in the β-globin (HBB) gene. 1 Whereas causative mutations may be corrected by precise gene correction based on homology-directed repair, imprecise disruption of genome elements by non-homologous end joining is inherently more efficient and more suitable for long-term repopulating cells. 2 This has already prompted the pursuit of disruption-based reactivation of the HBB paralog g-globin as a potentially universal genome-editing strategy to treat patients with β-hemoglobinopathies, 3 which is as yet unproven in the clinic. The common β-thalassemia allele IVSI-110 (HBB ) has an aberrant splice acceptor site that leads to abnormal splicing. 4 Here we investigated the use of a mutation-specific and disruption-based approach to correct HBB . Based on both transcription activator-like effector nucleases (TALEN) and CRISPR/Cas9 RNA-guided HBB -targeting nucleases we analyzed non-homologous end joining-based indel events at on-and off-target sites, and the efficiency of functional correction in patient-derived CD34 + -derived HBB IVS-110(G>A) -homozygous erythroblasts. Both platforms showed significant correction at the RNA, protein and morphological levels, with up to 95% on-target disruption, using a design that minimized d-globin (HBD) offtarget activity. The present study establishes suitable target sequences for effective restoration of normal splicing and validates gene disruption by virus-and DNA-free delivery of nucleases as potential therapy for HBB thalassemia.The HBB mutation resides 19 nucleotides upstream of the normal intron-1 splice acceptor site. We identified one CRISPR/Cas9 and two TALEN-pair target sites compatible with platform-specific sequence constraints, proximity of exon 2, and the need to discern HBB from HBD for therapy by disruption ( Figure 1, Online Supplementary Figure S1). Predicted double-stranded break sites were adjacent to the aberrant splice acceptor site for the RNA-guided nuclease (RGN) and upstream for TALEN pairs, TALEN R1/L1 (R1/L1) and TALEN