The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.
PLoS ONE (Impact Factor: 3.23). 09/2009; 4(9):e6995. DOI: 10.1371/journal.pone.0006995
Source: PubMed


Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the "core", which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5' core with a novel 3' 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.

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Available from: Fabrizia Urbinati, Oct 07, 2015
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    • "In order to incorporate the HS4 into RVs, several authors have minimized the size of the HS4 to 250 bp (HS4-Core), 400 bp (HS4-Ext) and 650 bp (HS4-650) which contain the main domains required for its insulator activity [15], [26], [27]. As observed by other authors [15], [26], [27], [32], [39], we observed a reduction in%rCV and silencing but also a general decrease on viral titer and transgene expression levels of all the SE-HS4 LVs. Nonetheless, a lower decrease on vector titer and transgene expression levels was observed for the HS4-650 element in comparison with the HS4-Core and the HS4-Ext. "
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    ABSTRACT: Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3' LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells.
    PLoS ONE 01/2014; 9(1):e84268. DOI:10.1371/journal.pone.0084268 · 3.23 Impact Factor
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    • "Novel and shorter elements derived from the cHS4 that confer enhancer-blocking activity do not require duplication in order to be effective, and do not decrease the viral titer. Therefore, they could be utilized for this purpose [32]. Furthermore, we observed that this vector was also effective in mice affected by β-thalassemia intermedia when administered via BMT of HSCs pretreated with the lentiviral construct, producing a phenotype more similar to that of WT mice transplanted with normal BM. "
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    ABSTRACT: Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.
    PLoS ONE 03/2012; 7(3):e32345. DOI:10.1371/journal.pone.0032345 · 3.23 Impact Factor
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    • "Miccioetal.,2008),althoughtheuseofthelatterisconsid- eredalmostnecessarybecausenotonlydotheyincrease expressionofb-globin(Arumugametal.,2007)buttheyalso havebeenproveninalargenumberofstudies(Despratand Bouhassira,2009;Hanawaetal.,2009)toactmoreefficiently inreducingtheriskofinsertionalmutagenesis.Inaddition, Arumugamandcolleagues(2009)showedthatthecorese- quenceofcHS4alonedoesnotinsulateviralvectorseffec- tivelyandthatboth3¢aswellas5¢sequencesareneededfor fullinsulatoractivity.Overall,thesedatademonstratethat moreexperimentsshouldbeperformedinordertoobtaina definitiveconclusionconcerningtheeffectsofinsulators. Furthermore,anotherkeypointwouldbetheassessmentof theefficacyoffullyinsulatedvectorswithshorterexpression cassettesversusnoninsulatedvectorswithlargerglobin cassettesintermsoftiter,transgeneexpression,andsafety,in viewoftheupcomingclinicaltrials. "
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    ABSTRACT: To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the (A)γ-globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of (A)γ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34(+) hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.
    Human gene therapy 08/2011; 23(1):15-31. DOI:10.1089/hum.2011.048 · 3.76 Impact Factor
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