The GATA1 -HS2 Enhancer Allows Persistent and Position- Independent Expression of a b-globin Transgene Annarita Miccio1,2, Valentina Poletti3, Francesca Tiboni1, Claudia Rossi1, Antonella Antonelli1, Fulvio Mavilio2,3, Giuliana Ferrari1,4* 1 H. San Raffaele-Telethon Institute for Gene Therapy (HSR-TIGET), Istituto Scientifico H. San Raffaele, Milan, Italy, 2 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy, 3 Laboratory of Gene Expression, Istituto Scientifico H. San Raffaele, Milan, Italy, 4 University Vita-Salute San Raffaele, Milan, Italy Abstract Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a b-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine b-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and b-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of b- globin gene transfer by reducing expression variegation and epigenetic silencing. Citation: Miccio A, Poletti V, Tiboni F, Rossi C, Antonelli A, et al. (2011) The GATA1-HS2 Enhancer Allows Persistent and Position-Independent Expression of a b- globin Transgene. PLoS ONE 6(12): e27955. doi:10.1371/journal.pone.0027955 Editor: Tadayuki Akagi, Kanazawa University, Japan Received September 20, 2011; Accepted October 28, 2011; Published December 2, 2011 Copyright: 2011 Miccio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Italian Telethon Foundation (Core Grant TIGET), the Italian Ministry of University and Research (PRIN 2008), and the European Commission (FP7, PERSIST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: giuliana.ferrari@hsr.it Introduction b-thalassemias are a group of autosomal recessive disorders characterized by reduced or absent synthesis of hemoglobin b- chains. Allogeneic bone marrow (BM) transplantation from HLA-matched related donors provides a definitive cure for this disease [1]. Gene therapy, i.e., transplantation of autologous, genetically corrected hematopoietic stem cells (HSCs), is a potential alternative for patients lacking a compatible donor. Gene therapy of b-thalassemia requires efficient transfer and appropriately regulated expression of a b-globin transgene. Lentiviral vectors (LV) carrying a human b-globin gene under the control of its minimal promoter and elements of the ß-globin locus-control region (LCR) transduce repopulating HSCs and allow long-term correction of the murine and human b- thalassemia phenotype in pre-clinical models [2,3,4,5,6]. Limited therapeutic efficacy has been recently observed in one patient treated with transduced CD34+ human hematopoietic stem/ progenitor cells (HPCs) [7]. A significant limitation of existing b-globin vectors is variegation and epigenetic silencing of transgene expression, which the activity of the b-globin LCR is not sufficient to overcome [8]. Several attempts have been undertaken to reduce chromatin-mediated silencing, such as the introduction of insulators or scaffold/matrix- attachment regions (S/MARs) in the vector backbone. Insertion of a 1.2-kb insulator derived from a chicken a-globin DNase hypersensitive site (cHS4) in the viral long terminal repeats (LTRs) led to complete correction of the thalassemic phenotype in the erythroblastic progeny of transduced human HPCs in vitro and in vivo [4,9]. Insertion of the cHS4 in the LTRs has, however, detrimental effects on the vector titer, which is partially overcome by reducing the insulator size [10,11]. A 573-bp insulator isolated from the sea urchin arylsulfatase locus (ArsI) was reported to protect LV from silencing by maintaining an active chromatin structure in an orientation-dependent manner, though not in all cell types or differentiation stages [12,13]. Another sea urchin chromatin insulator, sns5, was shown to protect a gamma- retroviral vector from the negative influence of chromatin in a mouse erythroid cell line [14]. Addition of S/MARs from the human b-interferon or immunoglobulin heavy chain loci, alone or in combination with insulators and enhancers, reduces silencing and provides position-independent transgene expression in hematopoietic cell lines and primary cells [15,16,17]. More recently, a ubiquitously acting chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus has been successfully used to minimize the influences of neighboring chromatin on single-copy transgene expression in hematopoietic cells [18,19]. An alternative strategy to protect an integrated expression cassette from the effect of the surrounding chromatin is the use of transcriptional enhancers, such as the metallothionein or a-globin HS40 enhancer or the DNase hypersensitive site II (HS2) of the PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e27955