Figure 4. A low G-GLOBE vector copy number is sufficient to correct of the murine thalassemic phenotype. (A) Hb concentra- tion in peripheral blood of mice transplanted with mock-transduced th3/+ (white column; n = 8), normal (WT; black column; n = 9) and GLOBE- (GLOBE; light gray column; n = 8) and G-GLOBE (G-GLOBE; dark gray column; n = 8) transduced BM th3/+ cells. Values represent the mean and SEM for each group of animals. Hb levels were not significantly different in th3/+ mice transplanted with GLOBE- and G- GLOBE-transduced HSCs. (B) Percentages of Ter119+ cells in BM of mice transplanted with mock-transduced th3/+ (white column; n = 8), normal (WT; black column; n = 9) and GLOBE- (GLOBE; light gray column; n = 8) and G-GLOBE (G-GLOBE; dark gray column; n = 8) transduced BM th3/+ cells. Values represent the mean and SEM for each group of animals. Persistent Expression of a b-Globin Transgene The proportion of Ter119+ erythroid cells were not significantly different in th3/+ mice transplanted with GLOBE- and G-GLOBE-transduced HSCs. (C) Lack of selection of erythroid progenitors carrying multiple copies of G-GLOBE. The Ter119+ fraction was sorted from the non-erythroid (Ter1192) sub-population of BM cells from mice transplanted with GLOBE-, G-GLOBE- and LV GFP-transduced cells and VCN was determined by qPCR. The proportion of GLOBE-transduced cells in the Ter119+ fraction was 2.5 fold higher than that observed in the Ter1192 compartment, indicating an in vivo selective advantage of highly transduced erythroid progenitors [5] (**P,0.01). In marked contrast, no statistically significant difference was observed for mice receiving G-GLOBE- and GFP-transduced BM cells (P.0.1), thus indicating 1 copy of G-GLOBE per cell is sufficient to produce normal Hb levels (Figure 4A). Interestingly, G GLOBE is able to rescue the thalassemic phenotype with a significantly lower VCN compared to GLOBE (*P,0.05). doi:10.1371/journal.pone.0027955.g004 background only in G-GLOBE-transduced cells. GATA1 and CBP occupancy at the endogenous b-globin LCR HS4 element, chosen as an internal control, was similar in GLOBE and G- GLOBE-transduced cells (Figure S6). We then measured histone acetylation levels at different regions in the GLOBE and G-GLOBE proviruses in transduced HEL cells. Primers specific for the 59LTR and the b-globin promoter (b-promoter-HS2) were used to amplify ChIP products by qPCR (Figures 7A). Chromatin was immunoprecipitated using anti- bodies against pan-acetylated H3 and specific acetylated lysines of H3 and H4 (H3K18, H4K5 and H4K8), which are targets of CBP activity. The G-GLOBE provirus exhibited higher H3 and H4 acetylation levels at the 59LTR compared to the GLOBE provirus (Figure 7B). This difference is maintained at the b-globin promoter region (Figure 7C), suggesting the spreading of acetylated histone marks along the chromatin fiber. Histone acetylation levels of the control, transcriptionally active Aldolase A gene were comparable in both GLOBE- and G-GLOBE- transduced cells (Figure S7). Concomitantly, the H3K4 trimethy- lation (H3K4me3), typical of active promoters, tended to be higher at the 59 LTR of G-GLOBE compared to GLOBE, whereas two heterochromatin marks (H3K9me3 and H3K27me3) were reduced (Figure S8A and S8B). H3K4me1, which marks enhancer regions [41], was increased at the GATA1-HS2- containing 59LTR (Figure S8B). Overall, these data indicate that GATA1, together with CBP, maintains an open chromatin structure by increasing the histone acetylation levels in the chromatin domain delimited by the GATA1-HS2-modified LTRs. Discussion Gene therapy of thalassemia requires vectors expressing high, persistent and regulated levels of b-globin. The use of b-globin promoters, enhancers, and reduced versions of the LCR allows the expression of potentially therapeutic levels of b-globin in the context of LV, as demonstrated in human thalassemic erythro- blasts in vitro [6,9] and in murine models of thalassemia in vivo [2,5,42,43]. LV integrate in different sites in the genome, and although they prefer open and transcribed chromatin regions [44], their expression is susceptible to chromosomal position effects leading to transgene silencing or variegated expression [2,8,45]. The reduced versions of the b-globin LCR are apparently insufficient to overcome position-dependent variability of gene expression in erythroblasts. Chromatin insulators, such as the cHS4 element, reduce only partially positional effects, while affecting both titer and genetic stability of the vectors [7,10,11]. The outcome of the only patient treated so far with a b-globin LV indicates a limited contribution of the vector-derived protein in the PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e27955