Integrated vector and experimental design. (A) The BGI vector was constructed using an SIN lentiviral backbone containing a 1.2-kb cHS4 insulator (cHS4-I) element inserted to replace the 398-bp U3 promoter and/or enhancer deletion (U3Δ). Upon proviral integration into host genome, the U3 region containing the cHS4 is copied over to the 5′LTR. A 3.1-kb human β-globin LCR region consisting of hypersensitive sites (HS) 2, 3, and 4; β-globin promoter (βPr; 254 bp); the β-globin gene (with a 372-bp deletion in IVS2); and the 3′ enhancer (3′E) were cloned in reverse orientation to the viral transcriptional unit downstream of the rev response element (RRE) and the central polypurine tract (cPPT). (B) Experimental design. Cryopreserved normal and thalassemia major bone marrow CD34⁺ cells were thawed simultaneously and transduced twice in the presence of recombinant human IL-6, stem cell factor, Flt-3 ligand, and thrombopoietin (6/S/F/T) within 24 hours. Cells were washed and portions of cells were plated in vitro, in erythroid liquid cultures, in semisolid medium for colony-forming assays, and transplanted into β2mnull NOD-SCID mice. Arrows depict serial time points at which various analyses were performed.