Schematic representation of the 2 novel lentiviral vectors for SCID-X1 gene therapy and their activity in various cell types. (A) Both vectors are based on third-generation lentiviral vector backbone. The enhancer/promoter in the long-terminal repeat region was deleted (ΔU3) and replaced with a 400-bp chromatin insulator element (Ins) from the chicken β-globin locus. The CL20i4-hγc-Revgen vector contains the entire human IL2RG gene (γc genomic), including all 8 exons, 7 introns, and the 1.2-kb promoter (γcPro), in reverse genomic orientation relative to the vector backbone. The CL20i4-EF1α-hγcOPT vector contains codon-optimized human γc cDNA (hγc), the expression of which is driven by a 233-bp human elongation factor α-promoter (EF1α). Ψ indicates viral packaging signal; cpPT, the central polypurine tract; RRE, lentiviral Rev Response Element; MSCV-hγc vector, γ-retrovirus vector expressing human γc cDNA and an IRES-GFP cassette; and IRES, internal ribosome entry site. (B) Epstein-Barr virus–transformed human B lymphoblast from an SCID-X1 patient (EBV-B^SCID-X1 cells) and human epithelial cervical cancer cell line HeLa were transduced with either the Revgen vector or the EF1α vector. Two weeks later, cell-surface expressions of γc were measured by flow cytometry after staining with anti-CD132 monoclonal antibody. EBV-B^WT cells indicates Epstein-Barr virus–transformed human B lymphoblasts from a healthy donor; MFI, mean fluorescence intensity of the gated subpopulation; and VCN, average vector copy number in the entire population.