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.

Keywords: CGD; Clinical Immunology; Genetherapy; Hematology; IPSC; Immunology; Neutrophils; Silencing.

Figure 1.. Gene therapy of iPSC and differentiation to neutrophils.

(A) Workflow of iPSC Work. Methylene blue/eosin staining shows that early yield of neutrophil factories contain cells with band cell morphology (neutrophil progenitors), whereas late yield of the factory contains cells with mature neutrophil morphology (with the neutrophil typical multi-lobal segmented nuclei) and also macrophage morphology (with single round nuclei). (B) p47 expression in OCT_3/4 gated iPSC over time. P47 expression of UCOE_ SF_p47 is stable over time, but decreases in SF_p47 transduced cells. (C) DNA methylation analysis in iPSC after 20 days of culture. CpG dinucleotides in individual subcloned PCR products are shown in horizontal orientation with black/white circles indicating methylated/unmethylated CpGs. UCOE protects the SFFV promoter from methylation, whereas unprotected SFFV promoter is strongly methylated in iPSC 20 days after transfection. (D) Gating strategy and p47 expression in yield of neutrophil factory day 68 after transduction. p47 expression of UCOE_ SF_p47 is maintained at high level in neutrophils (FCSlow (gate A), CD15+CD14- (gate N)) and in macrophages (FCSlow, CD14+CD15- (gate M)), but significantly reduced in SF_p47 transduced cells in comparison to p47phox expression in iPSC on day 6 (Fig. 1B).,(E) Detection of ROS production in differentiated CD15+ neutrophils in a dihydrorho-damine 123 assay. ROS production leads to increased fluorescence resulting in a shift to the right. Percentage of ROS producing neutrophils is high for UCOE_ SF_p47, but marginal in SF_p47 in comparison to p47phox expression in iPSC on day 6 (Fig. 1B).