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A recent gamma-retroviral clinical Chronic Granulomatous Disease (CGD) gene therapy (GT) trial achieved proof-of-concept but was accompanied by activation of oncogenes and transgene silencing. The ubiquitous chromatin opening element (UCOE) comprises the sequences of two divergently oriented house-keeping gene promoters and is known to have anti-silencing properties. In a screen we identified two novel UCOE constructs that prevent adjacent promoter methylation in P19 cells. Experiments were continued with the shorter UCOE constructs in induced pluripotent stem cells (iPSC) derived from a p47phox-deficient CGD patient. The iPSC line was transduced with the lentiviral GT vectors expressing P47 under the constitutively active SFFV promoter with UCOE element (UCOE_SF) and without UCOE element (SF) adjacent to the SFFV promoter. The iPSC were expanded before propagation towards neutrophils. 20 days after transduction the UCOE_SF vector was protected from methylation in iPSC as previously shown in P19 cells, whereas the SF vector was heavily methylated in iPSC. The UCOE_SF vector maintained stable transgene expression in iPSC, macrophages and neutrophils, whereas the SF vector was strongly silenced. The UCOE_SF vector stably restored ROS production in neutrophils, whereas for the SF vector the count of ROS producing cells was marginal. To conclude, we have shown that the prevention of transgene silencing by UCOE is functionally and mechanistically preserved upon terminal neutrophil differentiation.

Original publication

DOI

10.19185/matters.201805000005

Type

Journal article

Journal

Matters

Publication Date

23/06/2019

Volume

2018

Addresses

Systems and Cell Biology of Neurodegeneration, University of Zurich, James Martin Stem Cell Facility, University of Oxford, Division of Immunology, University Children's Hospital Zurich, Div. of Immunology, University Children's Hospital Zurich, Children's Research Center, Associated Group Institute for Regenerative Medicine, Department of Psychiatry, Sir William Dunn School of Pathology, University of Oxford, Icahn School of Medicine at Mount Sinai, HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA, James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, Professor emeritus University Children's Hospital Zürich, Division of Immunology, Professor emeritus Children's Research Center, Div. of Immunology, University Children's Hospital Zurich, Children's Research Center, Associated Group Institute for Regenerative Medicine, University of Zürich, Center for Applied Biotechnology and Molecular Medicine, University of Zürich, Div. of Immunology, University Children's Hospital Zürich, Children's Research Center, Associated Group Institute for Regenerative Medicine, University of Zürich.