b-globin LCR, which relieve position effects at integration sites in cultured cells [20,21,22,23]. In the class of erythroid-specific enhancers, the GATA1-HS2, is a 200-bp element from the gene encoding GATA1 [24], a transcriptional activator that operates as a general switching factor for erythroid development [25]. GATA1 binds to the consensus sequence WGATAR within globin LCRs and regulatory regions of erythroid-specific genes and interacts with CBP, a histone acetyltransferase (HAT) that stimulates GATA1 transcriptional activity [26] by inducing an open chromatin configuration. CBP can directly acetylate GATA1 and, consequently, facilitates GATA1 chromatin occupancy [27,28]. The HS2 element contains a high affinity, palindromic GATA1 binding element that mediates positive autoregulation of GATA1 expression [29,30] and binds the transcription factors CP2 [31] and BCL11A [32]. In this study, we tested the ability of GATA1-HS2 in counteracting repressive chromatin effects in the context of LV-delivered transgenes. The element was inserted into the LTRs of a LV expressing a GFP gene under the control of the silencing-prone cytomegalovirus (CMV) promoter or a b-globin minigene under the control of a reduced-size LCR. The activity of these vectors was tested in the progeny of transduced, repopulating murine HSCs and human CD34+ cells. We show that the GATA1-HS2 is able to increase the probability of long- term transgene expression, and to rescue the thalassemic phenotype at low copy number in a murine model of the disease. Chromatin immunoprecipitation (ChIP) studies showed that GATA1 and CBP bind GATA1-HS2-containing LTRs and induce hyperacetylation at specific histone residues throughout the provirus. These results show that an effective approach to shield integrated transgenes from negative chromosomal position effects is represented by the inclusion in LV LTRs of sequences co-targeted by transcription factors and chromatin modifying enzymes. Results A strategy to achieve persistent and position- independent transgene expression in erythroblastic cells We previously reported that the GATA1-HS2 element cloned in transcriptionally active viral LTR drives erythroid-restricted transgene expression in both MLV- and HIV-derived vectors [33,34]. To test whether GATA1-HS2 may positively influence the long-term transgene expression in erythroblastic cells, a previously described [35] LV expressing GFP from the CMV promoter (CMV-GFP, Figure 1A) was modified by cloning the 200-bp HS2 element in the 39 LTR, to obtain the G-CMV-GFP vector (Figure 1B). The CMV promoter was chosen because of its high susceptibility to silencing in vitro and in vivo [18]. The activity of the CMV-GFP and G-CMV-GFP vectors was comparatively ana- lyzed following transduction of murine HSCs and human cord blood-derived, CD34+ cells. The GATA1-HS2 element protects the CMV promoter from silencing in the erythroid progeny of murine HSCs BM cells from CD45.1 C57BL/6 (C57BL/6-Ly-5.1) mice were transduced with either the CMV-GFP or the G-CMV-GFP vector and transplanted in lethally-irradiated CD45.2 co-isogenic mice ((C57BL/6; n = 9 and n = 8, respectively). Transduction efficiency, calculated as percentage of GFP+ colony forming cells (CFCs) in methylcellulose assay, averaged 75% for both vectors (data not shown). Engraftment of donor cells, as proportion of CD45.1+ to total CD45+ cells, ranged from 88 to 99% at 12 months after transplantation. BM from recipient Persistent Expression of a b-Globin Transgene animals was analyzed for GFP expression in the erythroid (Ter119+) and non-erythroid (Ter1192) progeny of transduced, long-term repopulating HSCs. The proportion of Ter119bright cells was comparable in both groups (3462% and 3662%, for CMV-GFP and G-CMV-GFP, respectively). GFP+ cells were detected at a significantly higher proportion in the Ter119+ compartment of mice transplanted with G-CMV-GFP-trans- duced HSCs, compared to mice transplanted with CMV-GFP- transduced HSCs (5664% vs. 2868%, P,0.05, Figure 2A and Figure S1A). The mean fluorescence intensity (MFI) of Ter119+/GFP+ cells was also significantly higher in mice transplanted with G-CMV-GFP-transduced cells (208625 vs. 96610, P,0.001, Figure 2A and Figure S1B). Conversely, the proportion of GFP+ cells and the MFI in the Ter1192, myeloid and lymphoid compartments were comparable in the two groups (29611% vs. 3265%: MFI: 372689 vs. 248623, Figure 2A). The average vector copy number (VCN) in total BM cells, analyzed by qPCR, was comparable in both groups (3.560.9 for CMV-GFP vs. 3.060.6 for G-CMV-GFP, P.0.1). To analyze gene expression in the clonal progeny of long-term HSCs, BM from primary transplanted animals (3 different donors for each vector; Table S1) was transplanted in secondary CD45.2 C57BL/6 recipients (n = 8 per group, Table S1). A total of 66 12-day colonies derived from spleen colony-forming unit (CFU-S) cells were isolated and analyzed by PCR for the presence of the provirus, and by cytofluorimetry for Ter119 and GFP expression. The proportion of Ter119bright cells was comparable in both groups (data not shown). We observed a .2-fold increase in the percentage of GFP+ colonies carrying the G-CMV-GFP vector compared to those carrying the CMV-GFP vector (85% vs 40%; P,0.001, Figure 2B). The average VCN detected by qPCR analysis was 1.4 and 1.8 for G-CMV-GFP and CMV-GFP colonies, respectively (not shown). These data indicate that the HS2 element significantly increases the probability of transgene expression in the clonal erythroid progeny of murine repopulating HSCs by reducing stable and/ or variegating position effect. The GATA1-HS2 element maintains high level of transgene expression in the erythroid progeny of human HPCs in vitro and in vivo We then tested the activity of CMV-GFP and G-CMV-GFP vectors in human cord blood-derived CD34+ cells. To analyze transgene expression at clonal level, CMV-GFP- and G-CMV- GFP-transduced CD34+ cells were cultured in methylcellulose for 14 days, and single colonies were scored for GFP expression by fluorescence microscopy inspection, and analyzed for the presence of vector sequences by PCR. The vast majority (90%) of cells from BFU-Es (erythroid burst-forming units) carrying the G-CMV-GFP vector expressed the transgene compared to a significantly lower proportion (63%) of those harboring the CMV-GFP vector (P,0.005, Figure 3A), while no significant difference was observed in the proportion of GFP+ cells from CFU-GM (granulocyte-macrophage colony-forming units) between the two groups (75% vs. 68%, P.0.1, Figure 3B). Transduced HPCs were then assayed in vivo after transplanta- tion in sub-lethally irradiated NOD-SCID mice (n = 5 and n = 4 for the CMV-GFP and G-CMV-GFP vector). Transduction efficiency ranged from 70 to 80% for both vectors. Eight to 11 weeks after transplantation, the engraftment of SCID-repopulating cells (SRCs) ranged from 8 to 28%, as determined by the proportion of human CD45+ cells in the mouse BM. Staining with antibodies against lineage markers (CD19, CD13, and CD34) showed the presence of human lymphoid, myeloid, and PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e27955