Donor IDLV for HDR were generated as described. ZFNs targeting intron 1 of PPP1R12C or exon 5 of IL2RG were expressed by mRNA electroporation. CD34+ cells from human umbilical cord blood or bone marrow were used on approval by the San Raffaele Hospital Bioethical Committee, stimulated in serum-free medium with early acting cytokines, infected with IDLVs at a multiplicity of infection (MOI) 100–500, and then electroporated with 175 mg/ml ZFNs encoding mRNAs (P3 Primary Cell 4D-Nucleofector X Kit, program EO-100; Lonza). Targeted integration was assessed by PCR and Southern blot while ZFN activity was determined by Cel1 assay and deep sequencing of genomic target sites. The treated CD34+ cells at day 4 of culture were infused intravenously into sublethally irradiated 8–11-week-old NOD-SCID-Il2rg-/- (NSG) mice. To expand human T and NK cells, 4 million MDA3-MB231 tumour cells expressing human IL-7, IL-15 and GM-CSF were implanted orthotopically in the mammary fat pad of NSG mice 14 weeks after CD34+ cells transplantation. Functional assays on IL2RG-edited T cells collected from transplanted mice were carried out after stimulation with beads conjugated to anti-human CD3 and CD28 antibodies and sorting for GFP expression. Summary (Lonza): The authors optimized a protocol that allows gene repair in repopulating human haematopoietic stem cells (HSCs), key target cells for treating blood inherited disorders. Genovese et al. achieved efficient integration of a GFP sequence into the genome of human HSCs at chosen sites, including a protein-coding region (exon 5) of the IL2RG gene that is mutated in patients with X-linked severe combined immunodeficiency (SCID-X1). HSCs were pre-stimulated with cytokines, delivered with the recombination template, and followed by a transfection with Zn-finger nuclease mRNA using the 4D-Nucleofector. Cells were prevented from differentiation using dmPGE2 and SR1 factors. A portion of the cells was modified with success, then transferred into immunodeficient mice. Edited functional cells could be detected for up to 18 weeks and further transferred into a second recipient. The authors were also able to correct HSC derived from a patient carrying Il2rg-/- SCID-X1 syndrome. Using this approach, the authors opened new avenues for treating SCID-X1 disease and other genetic diseases using genome editing tools.