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Lentiviral gene therapy vector with UCOE stably restores function in iPSC-derived neutrophils of a CDG patient


Haenseler, Walther; Kouzmenko, Elena; Brown, Cathy; Smalls-Mantey, Adjoa; Trasher, Adrian; James, William; Seger, Reinhard; Reichenbach, Janine; Cowley, Sally A; Siler, Ulrich (2018). Lentiviral gene therapy vector with UCOE stably restores function in iPSC-derived neutrophils of a CDG patient. Matters:1-7.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:23 May 2018
Deposited On:05 Feb 2019 20:45
Last Modified:29 Jul 2020 09:12
Publisher:ScienceMatters AG
ISSN:2297-8240
OA Status:Hybrid
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.19185/matters.201805000005
Related URLs:https://sciencematters.io/articles/201805000005 (Author)
https://sciencematters.io/ (Publisher)
PubMed ID:31008103
Project Information:
  • : FunderFP7
  • : Grant ID305011
  • : Project TitleGene Therapy for X-linked Chronic Granulomatous Disease (CGD)
  • : FunderFP7
  • : Grant ID261387
  • : Project TitleAdvanced Cell-based Therapies for the treatment of Primary ImmunoDeficiency
  • : FunderSNSF
  • : Grant ID320030-121983
  • : Project TitleDevelopment of a gammretroviral gene therapy vector for the p47phox deficient form of CGD for later clinical use
  • : FunderSNSF
  • : Grant IDP2ZHP3_148607
  • : Project TitleThe role of microglia in alpha-synuclein related loss of dopaminergic neurons in Parkinson's disease

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