Williams, S, Mustoe, T, Mulcahy, T, Griffiths, M, Simpson, D, Antoniou, M et al.. CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells. BMC Biotechnol 5: 17

ICL, Londinium, England, United Kingdom
BMC Biotechnology (Impact Factor: 2.03). 02/2005; 5(1):17. DOI: 10.1186/1472-6750-5-17
Source: PubMed


The hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing.
In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression.
Vectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.

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Available from: Michael N Antoniou, May 09, 2014
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    • ") and β-thalassemia major (Pawliuk et al., 2001; Persons et al., 2003; Puthenveetil et al., 2004) has been achieved using lentivirus vectors that contain complex regulatory sequences from the LCR region. Recent advances in vector design have improved gene transfer for the hemoglobinopathies such as the ubiquitous chromatin opening element (UCOE) augmented spleen focus forming virus (SFFV) promoter/enhancer which provides lentivirus vectors with a natural tropism for the hematopoietic system (Antoniou et al., 2003; Williams et al., 2005) resulting in reproducible and stable function in BM and all differentiated peripheral hematopoietic cell lineages (Zhang et al., 2007). "
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    ABSTRACT: Congenital diseases are responsible for over a third of all pediatric hospital admissions. Advances in prenatal screening and molecular diagnosis have allowed the detection of many life-threatening genetic diseases early in gestation. In utero transplantation (IUT) with stem cells could cure affected fetuses but so far in humans, successful IUT using allogeneic hematopoietic stem cells (HSCs), has been limited to fetuses with severe immunologic defects and more recently IUT with allogeneic mesenchymal stem cell transplantation, has improved phenotype in osteogenesis imperfecta. The options of preemptive treatment of congenital diseases in utero by stem cell or gene therapy changes the perspective of congenital diseases since it may avoid the need for postnatal treatment and reduce future costs. Amniotic fluid stem (AFS) cells have been isolated and characterized in human, mice, rodents, rabbit, and sheep and are a potential source of cells for therapeutic applications in disorders for treatment prenatally or postnatally. Gene transfer to the cells with long-term transgenic protein expression is feasible. Recently, pre-clinical autologous transplantation of transduced cells has been achieved in fetal sheep using minimally invasive ultrasound guided injection techniques. Clinically relevant levels of transgenic protein were expressed in the blood of transplanted lambs for at least 6 months. The cells have also demonstrated the potential of repair in a range of pre-clinical disease models such as neurological disorders, tracheal repair, bladder injury, and diaphragmatic hernia repair in neonates or adults. These results have been encouraging, and bring personalized tissue engineering for prenatal treatment of genetic disorders closer to the clinic.
    Full-text · Article · Dec 2014 · Frontiers in Pharmacology
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    • "The best characterized of these elements is derived from the human HNRPA2B1-CBX3 locus, which comprises dual divergently transcribed promoters driving the expression of the heterogeneous ribonucleoprotein A2/B1(HNRP2AB1) and the chromobox homolog 3(CBX3) housekeeping genes [10]. A 1.5 kb sequence derived from this locus, the A2UCOE, has convincingly been demonstrated to prevent promoter silencing and positional effect variegation in vitro and in in vivo transplantation models when placed upstream of internal promoters in the SIN lentiviral configuration [9] [11] [12]. Furthermore, the A2UCOE was demonstrated to sustain promoter specificity in this situation, thus introducing the element as an elegant choice to achieve reliable long-term transgene expression in gene therapy approaches [13] [14]. "
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    ABSTRACT: Notwithstanding recent successes, insertional mutagenesis as well as silencing and variegation of transgene expression still represent considerable obstacles to hematopoietic gene therapy. This also applies to O(6)-methylguanine DNA methyltransferase (MGMT)-mediated myeloprotection, a concept recently proven clinically effective in the context of glioblastoma therapy. To improve on this situation we here evaluate a SIN-lentiviral vector expressing the MGMT(P140K)-cDNA from a combined A2UCOE/PGK-promoter. In a murine in vivo chemoselection model the A2UCOE.PGK.MGMT construct allowed for significant myeloprotection as well as robust and stable selection of transgenic hematopoietic cells. In contrast, only transient enrichment and severe myelotoxicity was observed for a PGK.MGMT control vector. Selection of A2UCOE.PGK.MGMT-transduced myeloid and lymphoid mature and progenitor cells was demonstrated in the peripheral blood, bone marrow, spleen, and thymus. Unlike the PGK and SFFV promoters used as controls, the A2UCOE.PGK promoter allowed for sustained vector copy number-related transgene expression throughout the experiment indicating an increased resistance to silencing, which was further confirmed by CpG methylation studies of the PGK promoter. Thus, our data support a potential role of the A2UCOE.PGK.MGMT-vector in future MGMT-based myeloprotection and chemoselection strategies, and underlines the suitability of the A2UCOE element to stabilize lentiviral transgene expression in hematopoietic gene therapy.
    Full-text · Article · May 2014 · Biomaterials
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    • "The UCOE spanning the dual, divergently transcribed promoters of the human TBP-PSMB1 and HNRPA2B1-CBX3 housekeeping genes was shown to mediate stable expression of transgenes even when integrated in heterochromatic regions and reduce variegation effects.14 In combination with the immediate early promoter/enhancer of the human cytomegalovirus (hCMV), the UCOE derived from the HNRPA2B1-CBX3 locus (A2UCOE) mediates improved levels of expression and proportion of cells expressing the transgene at detectable levels.15 Another cis-acting element we examined is the 5′-HS4 chicken-β-globin insulator (cHS4), which has been considered as an ideal candidate for use in gene transfer applications due to its barrier and enhancer-blocking function.16 "
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    ABSTRACT: The vector pEPI was the first nonviral and episomally replicating vector. Its functional element is an expression unit linked to a chromosomal scaffold/matrix attached region (S/MAR). The vector replicates autonomously with low copy number in various cell lines, is mitotically stable in the absence of selection over hundreds of generations, and was successfully used for the efficient generation of genetically modified pigs. Since it is assumed that establishment of the vector is a stochastic event and strongly depends on the nuclear compartment it reaches after transfection, it is of great interest to identify genomic sequences that guide DNA sequences into certain nuclear compartments. Here we inserted genomic cis-acting sequences into pEPI and examined their impact on transgene expression, long-term stability, and vector establishment. We demonstrated that a ubiquitous chromatin-opening element (UCOE) mediated enhanced transgene expression, while an insulator sequence (cHS4) increased establishment efficiency, presumably via an additional interaction with the nuclear matrix. Thus, besides being a promising alternative to currently used viral vectors in gene therapeutic approaches, pEPI may also serve as a tool to study nuclear compartmentalization; identification of genomic cis-acting sequences that are involved in nuclear organization will contribute to our understanding of the interplay between transgene expression, plasmid establishment, and nuclear architecture.Molecular Therapy-Nucleic Acids (2013) 2, e118; doi:10.1038/mtna.2013.47; published online 3 September 2013.
    Full-text · Article · Sep 2013 · Molecular Therapy - Nucleic Acids
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