Persistent Expression of a b-Globin Transgene Figure 1. Structure of CMV-GFP, G-CMV-GFP, GLOBE, G-GLOBE and PGK-GFP lentiviral vectors. (A–B) Schematic map of the CMV-GFP and G-CMV-GFP vectors in their proviral form. In both vectors, the CMV promoter drives the expression of a GFP transgene. In the CMV-GFP vector, the GATA1-HS2 enhancer was inserted into the self-inactivated LTRs (sin-18 LTRs) carrying 400 bp deletion in HIV U3 region (G sin-18 LTR). The rev- responsive element (RRE), the splice donor site (SD), the splicing acceptor site (SA), the central polypurine tract (cPPT), the woodchuck post- regulatory element (WPRE) are shown. (C) Schematic representation of the b-globin expressing G-GLOBE. The sin-18 LTR, RRE, SD, SA, cPPT, the human b-globin gene, the 2265/+50 b-globin promoter (bp) and the human LCR Dnase I hypersensitive sites HS2 and HS3 are shown. (D) Schematic representation of the b-globin expressing G-GLOBE vector. The GATA1-HS2 element was inserted into the self-inactivated LTRs (G sin 218 LTR). (E) Structure of the PGK-GFP lentiviral vector. A GFP transgene is under the control of the human phospoglycerate kinase promoter (PGK). doi:10.1371/journal.pone.0027955.g001 undifferentiated progenitors, indicating multilineage differentia- tion of SRCs in both mouse groups (data not shown). Since human erythroid differentiation occurs at low level in NOD-SCID chimeras, we tested GFP expression in the erythroblastic progeny of transduced SRCs ex vivo, in clonal cultures preferentially supporting the growth of human progenitors. BFU-E colonies were scored for both GFP expression and the presence of proviral vectors. Overall, 33 out of 40 (83%) of the G-CMV-GFP- transduced colonies expressed the transgene, compared to 33 out of 53 (62%) in the case of CMV-GFP (P,0.05, Figure 3C). The percentage of transduced CFU-GM colonies expressing the transgene was comparable for the two vectors (77% and 75% for CMV-GFP and G-CMV-GFP, respectively; Figure 3D). These data show that that GATA1-HS2 partially protects a CMV- driven transgene from silencing in the erythroid progeny of human HPCs and SRCs. The GATA1-HS2 element improves correction of the murine b-thalassemia phenotype by low copy number of a b-globin vector We previously showed that the GLOBE LV, expressing a human b-globin gene under the control of the b-globin promoter and a reduced-size LCR (Figure 1C) is able to correct the murine b-thalassemia phenotype by providing a selective advantage to genetically corrected erythroblasts [5]. We therefore tested the effect of introducing a copy of GATA1-HS2 in both LTRs of the GLOBE vector (G-GLOBE, Figure 1D) on the expression of the b-globin gene in a thalassemic mouse model. We used heterozygous C57BL/6-Hbbth3 knock-out mice (th3/+) [36], which show a pathophysiology comparable to that of patients with severe ß-thalassemia intermedia, that is chronic anemia, anomalies in RBC size and shape, and ineffective erythropoiesis. We transplanted 16 lethally-irradiated CD45.2, th3/+ mice with PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e27955