SCID-X1 is caused by mutations in the IL2RG gene and results in severe defects in T, B, and NK-cell mediated immunity. Transduction of hematopoietic stem and progenitor cells with IL2RG-expressing gamma-retroviral vectors can be clinically effective but has also caused leukemia in several trials due to vector integrations that activate T-cell proto-oncogenes. One approach to eliminate these genotoxic integrations is to use genome editing techniques to insert an IL2RG transgene into a genomic safe harbor loci. The choice of a genomic safe harbor rather than the endogenous IL2RG locus offers several advantages including correction of a wide variety of mutations with a single therapeutic cassette, potentially higher editing efficiency in selected safe harbor sites, and the ability to adapt the specific nuclease reagents to other diseases and therapeutic templates. We are testing two different IL2RG-expression cassettes for targeting and expression into the AAVS1 locus. First, we generated a recombination template that contains a codon-optimized IL2RG cDNA driven by the short elongation factor alpha (EF1a) promoter based on clinical data with this cassette being transmitted via a lentiviral vector and showing immune correction in human SCID-X1 subjects in an ongoing trial at the NIH Clinical Center. We first tested whether a sufficient level of IL2RG expression can be achieved when a single copy of the EF1α-IL2RG-cDNA cassette was inserted into the AAVS1 locus in human ED7R T-cells. This transgene cassette was flanked by 500bp homologous regions from the AAVS1 locus and transfected into ED7R cells, along with a pair of TALEN endonucleases that cleave within the AAVS1 locus. Three weeks after transfection, a distinct 7% subpopulation of cells expressed IL2RG on the cell surface, which was subsequently sorted by flow cytometry. We analyzed 10 single cell subclones from this population and confirmed that homologous recombination and single allele targeting had occurred in each of these clones, demonstrating efficient homologous recombination at the AAVS1 site. Flow analysis showed that the EF1α-IL2RG-cDNA cassette was expressed at approximately the same level as that seen in cells transduced with a single copy of the clinical EF1α-IL2RG-cDNA lentiviral vector, verifying that the AAVS1 is permissive for therapeutic expression levels at the single copy level. One potential disadvantage of the safe harbor approach is the potential loss of precise regulation of gene expression resulting from the use of such synthetic cDNA constructs. For this reason, we are also currently targeting the full 5.3 kb genomic IL2RG locus and endogenous promoter to the AAVS1 safe harbor to compare their IL2RG expression level with that obtained from the cDNA construct. We are also comparing editing efficiency at AAVS1 using several TALEN pairs as well as 4 Staphylococcus Aureus Cas9 guide RNAs, which are deliverable by “all-in-one” single stranded AAV6 vectors. To date, the best guide has led to 37 % allele editing in 293T cells when transfected as plasmid. We are currently producing AAV vectors that contain this guide RNA and will test this AAV vector, along with IDLV vectors that will transfer either the cDNA or genomic IL2RG templates, for editing efficiency and IL2RG gene expression in ED7R and human CD34+ cells.