Dataset Information

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

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.

SUBMITTER: Haenseler W

PROVIDER: S-EPMC6472893 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Haenseler Walther W Kuzmenko Elena E Smalls-Mantey Adjoa A Browne Cathy C Seger Reinhard R James William S WS Cowley Sally A SA Reichenbach Janine J Siler Ulrich U

Matters 20180523

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 <i>ubiquitous chromatin opening element</i> (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 P<sub>19</sub> cells. Exp ...[more]

PMID: 31008103

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Project description:BackgroundHIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells.ResultsWe have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency.ConclusionAs with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.

Project description:Tau aggregation within neurons is a critical feature of Alzheimer's disease (AD) and related tauopathies. It is believed that soluble pathologic tau species seed the formation of tau aggregates in a prion-like manner and propagate through connected neurons during the progression of disease. Both soluble and aggregated forms of tau are thought to have neurotoxic properties. In addition, different strains of misfolded tau may cause differential neurotoxicity. In this work, we present an accelerated human neuronal model of tau-induced neurotoxicity that incorporates both soluble tau species and tau aggregation. Using patient-derived induced pluripotent stem cell (iPSC) neurons expressing a tau aggregation biosensor, we develop a cell culture system that allows continuous assessment of both induced tau aggregation and neuronal viability at single-cell resolution for periods of >1 week. We show that exogenous tau "seed" uptake, as measured by tau repeat domain (TauRD) reporter aggregation, increases the risk for subsequent neuronal death in vitro These results are the first to directly visualize neuronal TauRD aggregation and subsequent cell death in single human iPSC neurons. Specific morphologic strains or patterns of TauRD aggregation are then identified and associated with differing neurotoxicity. Furthermore, we demonstrate that familial AD iPSC neurons expressing the PSEN1 L435F mutation exhibit accelerated TauRD aggregation kinetics and a tau strain propagation bias when compared with control iPSC neurons.SIGNIFICANCE STATEMENT Neuronal intracellular aggregation of the microtubule binding protein tau occurs in Alzheimer's disease and related neurodegenerative tauopathies. Tau aggregates are believed to spread from neuron to neuron via prion-like misfolded tau seeds. Our work develops a human neuronal live-imaging system to visualize seeded tau aggregation and tau-induced neurotoxicity within single neurons. Using an aggregation-sensing tau reporter, we find that neuronal uptake and propagation of tau seeds reduces subsequent survival. In addition, human induced pluripotent stem cell (iPSC) neurons carrying an Alzheimer's disease-causing mutation in presenilin-1 undergo tau seeding more rapidly than control iPSC neurons. However, they do not show subsequent differences in neuronal survival. Finally, specific morphologies of tau aggregates are associated with increased neurotoxicity.

Dataset Information

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

Publications

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

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