transgenes delivered to HSCs and HPCs by a LV. Introduction of GATA1-HS2 in the viral LTRs effectively protects from silencing an expression cassette driven by a silencing-prone CMV promoter in erythroblasts differentiating from transduced murine HSCs and human CD34+ HPCs in vitro and in transplantation assays in vivo. The positive effect on transgene expression was not observed in the non-erythroblastic lineages, indicating that the presence of the erythroid-specific GATA1 transcription factor is essential for the activity of the GATA1-HS2 element. GATA1 is expressed also in megakaryocytes, eosinophils, and mast cells, which cannot be detected at high frequency in fresh BM cells. In vitro cell culture experiments will be necessary to unravel the potential GATA1-HS2 anti-silencing effect in these lineages. We then tested the activity of GATA1-HS2 in the LTRs of the GLOBE LV, containing a human b-globin gene under the control of its promoter and a minimal version (HS2+HS3) of the b-globin LCR [5]. The GLOBE vector is able to correct thalassemia in a murine pre-clinical model of gene therapy [5] and in the erythroblastic progeny of human thalassemic HPCs [6]. The therapeutic efficacy is achieved even at low dose of transduced HSCs by in vivo selection of transgene-expressing erythroblastic cells, possibly overcoming the negative influence of chromatin surrounding the LV proviruses. We observed that silencing of the b-globin transgene in the erythroid progeny of human HPCs and murine HSCs transduced with the G-GLOBE vector was significantly reduced compared to cells transduced with the GLOBE vector. As a consequence, the G-GLOBE vector allowed correction of murine thalassemia at a lower copy number and without inducing the in vivo selection observed with the GLOBE vector [5]. Therefore, reducing position-dependent variability of gene expression increases the vector’s therapeutic potential, providing disease correction at lower vector copy number. Remarkably, the LTR modification does not impair the viral titer, probably because of the limited size of the GATA1-HS2 (2.06109 vs. 1.46109 TU/ml for a typical preparation of GLOBE and G-GLOBE, respectively). The major difference between the two vectors was observed in the progeny of long-term repopulating HSCs, assayed as CFU-S in secondarily transplanted mice, indicating that the GATA1-HS2 provides a more effective long-term protection from silencing compared to to the LCR alone. However, position effect variegation, resulting in expression or silencing in different colonies from CFU-S harboring the same provirus, was observed for both vectors, indicating that GATA1-HS2 increases the probability of expression but is unable to completely prevent silencing in the long-term progeny of transduced HSCs. In order to understand the molecular basis of the protective effect of GATA1-HS2, we carried out ChIP studies on erythro- blastic cells transduced with the GLOBE or G-GLOBE vectors. A typical function of enhancers is to prevent the formation of repressive chromatin structure by controlling histone acetylation and methylation around promoters. Binding of GATA1 to erythroid-specific enhancers during development or differentiation is a key factor in the initiation and maintenance of active chromatin structures [46]. The interaction of GATA1 with CBP/ p300 suggests at least one mechanism by which HATs might be brought to specific sites [26],[47]. By modifying chromatin-bound histones or GATA1 itself [28], acetylases enhance transcriptional activity of erythroid-specific loci [27]. In this context, GATA1 is more an ‘‘architectural’’ factor than a simple transcriptional activator. The essential role of GATA1 and CBP for the formation of an erythroid-specific acetylation pattern that is permissive for high levels of gene expression has been reported at the LCR and the murine ß-major globin gene promoter [40,48]. Our study Persistent Expression of a b-Globin Transgene shows that binding of GATA1 and CBP to the GATA1-HS2 element correlates with increased CBP-mediated histone acetyla- tion (H3K18, H4K5, and H4K8) at the level of the LTRs and the internal promoter. Although GATA1 and CBP bind also the LCR elements and the b-globin promoter within the vector, the presence of the GATA1-HS2 element at both sides of the transgene apparently increases CBP-mediated histone acetylation and spreads the acetylated histone domain to a chromatin region that encompasses the entire provirus. This might create a more stable, inheritable active chromatin configuration that renders the integrated transgene resistant to silencing in a large proportion of integration sites. Our results suggest that GATA1 establishes an active epigenetic state across transgenes flanked by GATA1-HS2 elements in the context of LV. Therefore, this vector design represents a way to shield integrated transgenes from negative chromosomal position effects, improving the therapeutic potential of LV-mediated gene therapy approaches. Further studies addressing the impact of this lineage-specific enhancer on cellular transcriptional activity are necessary to assess the risk-benefit balance for potential clinical use. Materials and Methods Lentiviral vectors The CMV-GFP lentiviral vector was previously described [35]. To construct the G-CMV-GFP vector, an EcoRI/SacI fragment containing part of the 39 LTR of the lentiviral vector pHR2 [49] was subcloned into the PvuII site of the pUC19 plasmid. The plasmid was digested with EcoRV and PvuII, in order to generate a 2418 to 218 deletion (where +1 is the transcription start site) in the U3 region of the LTR, and ligated with a 200-bp BamH1 human genomic fragment (2856 to 2655) containing the GATA1- HS2 [24] to obtain a chimerical self inactivating (SIN) LTR. The EcoRI-SfiI fragment containing the modified 39 LTR was then introduced into the EcoRI-SfiI sites of the CMV-GFP backbone [50], to obtain the G-CMV-GFP vector. To generate the G- GLOBE vector, the EcoRI-SfiI HS2 SIN LTR fragment was introduced into the SpeI-SfiI sites of the GLOBE vector backbone. Viral vector stocks were prepared by transient co-transfection of HEK293T cells using a three-plasmid system as previously described [5]. HEK293T cells were kindly provided by Luigi Naldini. Transduction of stable cell lines HEL (human erythroleukemia) cell line was grown in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin/strepto- mycin and 2 mM L-glutamine, and transduced with viral stocks at a multiplicity of infection (MOI) of 20 in the presence of 8 mg/ml polybrene. HEL cells were purchased by ECACC (European Collection of Cell Cultures). Transduction and transplantation of murine HSCs and human HPCs BM cells were harvested from C57BL/6-Ly-5.1 mice (B/6.SJL- CD45a-Pep3b, Jackson Laboratories), infected with CMV-GFP and G-CMV-GFP vectors, and injected into 8-wk-old C57BL/6 mice (Charles River) as previously described [33]. BM cells from C57BL/6-Hbbth3 mice (Jackson Laboratories), were infected and transplanted into 8-wk-old C57BL/6-Ly-5.1 and C57BL/6- Hbbth3 mice, as previously described [5]. C57BL/6 or C57BL/ 6-Ly-5.1 mice were lethally irradiated (950 cGy) and secondarily transplanted with 105 total BM cells from primary transplanted animals. Spleen colony-forming units (CFU-S) were isolated at day 12 from the spleen of 8- to 12-wk-old secondarily transplanted PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e27955