Persistent Expression of a b-Globin Transgene Figure 2. Analysis of transgene expression in the progeny of murine HSCs transduced with CMV-GFP and G-CMV-GFP. (A) Representative FACS analysis of the BM sub-populations of mice transplanted with CMV-GFP-(mice #6 and #7) and G-CMV-GFP-(mice #14 and #15) transduced cells and sacrificed 12 months after BMT. The cells were stained with an antibody that recognizes the erythroid-specific marker Ter119. The presence of GATA1-HS2 into the LTRs (G-CMV-GFP vector) is able to sustain transgene expression in the Ter119+ BM erythroid compartment. (B) Single erytroid CFU-S were collected from the spleens of secondary recipients 12 days after transplantation with CMV-GFP and G CMV-GFP-containing BM cells (7 and 9 mice, respectively). Donor BM cells are derived from primary transplanted mice which have received CMV-GFP- and G-CMV-GFP- transduced BM cells (3 different donors for each vector). Vector-containing clones carrying a comparable number of proviral copies were analyzed by FACS for transgene expression. The percentages of GFP+ (light grey) and GFP2(dark grey) vector-positive CFU-S are represented(LV+). Transgene expression was observed in 40% of CMV-GFP-containing CFU-S, whereas the presence of GATA1-HS2 element into the LTRs was able to sustain GFP expression in 85% of G CMV-GFP-transduced CFU-S. The difference in the proportion of transgene expressing CFU-S between the two groups was statistically significant (P,0.001; Fisher exact test). doi:10.1371/journal.pone.0027955.g002 BM cells from co-isogenic CD45.1 th3/+ donors, transduced with either GLOBE or G-GLOBE (n = 8 per group). Control mice were transplanted with either wild-type cells (WT control, n = 9) or mock-transduced thalassemic cells (th3/+ control, n = 8). Donor chimerism was .90% at 9–12 months after transplantation in all mice. The extent of phenotypic correction was similar in mice transplanted with G-GLOBE and GLOBE-transduced BM: Hb level, ineffective erythropoiesis (evaluated as percentage of Ter119+ cells in BM), hematocrit, RBC and reticulocyte counts were equally improved compared to th3/+ controls (Figure 4A, 4B and Figure S2). Likewise, expression of human b-globin, as analyzed by FACS analysis and HPLC, was comparable in the two groups (Table S2). However, the average VCN in the G-GLOBE-transduced, Ter119+ cells was significantly lower than in GLOBE-transduced erythroblasts (1.0260.36 vs. 1.7360.40, P,0.05), while no difference was observed in the Ter1192, myeloid population between the two groups (0.7060.20 vs. 0.8360.30) (Figure 4C). No significant difference in the VCN of Ter119+ vs. Ter1192 cells was observed in th3/+ mice transplanted with HSCs transduced with a GFP-expressing vector (0.8160.34 vs. 0.7860.32; P.0.1, Figure 1E and Figure 4C). These results indicate that G-GLOBE is able to correct the murine thalassemia at an average VCN of 1 without inducing selection of erythroblasts harboring a higher number of proviruses, as observed for the GLOBE vector. The GATA1-HS2 element prevents silencing of a b-globin transgene in the progeny of long-term repopulating HSCs To test the effect of GATA1-HS2 on b-globin transgene expression in the clonal progeny of long-term repopulating HSCs, BM cells harboring 0.3–1 GLOBE and 0.2–1 G-GLOBE average vector copies per cell were harvested from primary transplanted mice (4 donors per group) 12 months after transplantation and injected in wild-type secondary recipients. We mapped 2 to 15 different predominant proviral integration sites in the total BM of mice in the GLOBE group and 3 to 15 in mice in the G-GLOBE group, as analyzed by LM-PCR and sequencing (Table S1). Individual colonies from 12-day secondary CFU-S were isolated from recipient mice and analyzed for the presence of the provirus and for b-globin expression. qPCR analysis revealed that all transduced colonies harbored a single proviral copy. Human b- globin synthesis, detected by FACS analysis, was observed in erythroblasts from 14 out of 24 G-GLOBE-containing splenic colonies (58%), compared to 10 out of 40 GLOBE-containing colonies (25%), indicating that GATA1-HS2 significantly increases (P,0.005) the probability of transgene expression in the progeny of long-term HSCs (Figure 5A). Interestingly, this effect was not apparent in primary colonies from 12-day CFU-S: 10 out of 12 (83%) colonies harboring GLOBE and 15 out of 17 (88%) colonies harboring G-GLOBE expressed human b-globin (P.0.1) (Figure 5B). The average VCN was 1.6 for GLOBE-transduced and 1.4 for G-GLOBE-transduced primary colonies. Proviral integration sites in secondary CFU-S-derived colonies were individually sequenced and mapped by LM-PCR. 5 out of 11 GLOBE and 10 out of 12 G-GLOBE integrations were associated to transgene expression, while the remaining proviruses were always silent (Table S3 and S4), showing that a G-GLOBE provirus is less dependent on its genomic position than a GLOBE provirus for transgene expression (45% vs. 83% of active proviruses, Figure 5C). Indeed, some integrations (3 G-GLOBE and 2 GLOBE), independently identified in different colonies, were variably active (Table S3 and S4), indicating that position effect variegation does occur in the progeny of long-term repopulating HSCs. The number of these events was too small to establish whether variegation is reduced by the presence of GATA1-HS2 in the LTRs. The integration pattern of the G-GLOBE vector is not biased by the presence of the GATA1-HS2 element in the LTRs We previously showed that the introduction of the MLV enhancer in the HIV LTR causes subtle changes in the integration pattern of LV [37]. To test whether the presence of the GATA1- HS2 element could bias LV integration, we analyzed the integration site distribution of the CMV-GFP and G-CMV-GFP vectors in the genome of human CD34+ HPCs. A total of 438 CMV-GFP and 305 G-CMV-GFP integration sites were mapped by LM-PCR, annotated as TSS-proximal, intragenic and intergenic, and their relative distribution compared to that of 9,974 random control sequences (Figure S3A). Both vectors integrate preferentially into genes (71.2% and 69.5% for CMV- GFP and G-CMV-GFP, respectively, compared to 36.1% in the control sequences, Figure S3A) with no preference for TSSs (Figure S3B). To correlate vector integration with gene activity, we determined the gene expression profile of CD34+ cells at the time of transduction by Affymetrix microarray analysis [6], and analyzed the expression level of the genes targeted by the vectors. As expected [38], both LV targeted preferentially active genes compared to random controls (Figure S4). Finally, we correlated vector integration sites and epigenetic modifications obtained from previously published ChIP-sequencing data sets [39]. The 61-kb regions flanking CMV-GFP and G-CMV-GFP integration sites were equally enriched in PolII binding sites and transcription- associated H3K4me1, H3K36me3, H4K20me1, H3K9me1, and H3K27me1 histone modifications compared to random sequenc- es, with no differences between CMV-GFP and G-CMV-GFP (Figure S5). Similarly, H3K4me3, H3K9me3 and H3K27me3 modifications and binding of the H2A.Z histone variant were under-represented around both LV integrations (Figure S5). The presence of GATA1-HS2 in the LTR has therefore no apparent influence on the selection of integration sites of the LV. PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e27955