Neutralisation of FX-binding deficient Ad5 vectors by naïve murine serum.

<p>(A) A549 and (B) SKOV3 cells: Ad5 and its derivatives (2x10¹⁰ vp/mL) were incubated with RPMI-1640 media, 90% C57BL/6 mouse serum or 1–90% C57BL/6 mouse serum in RPMI-1640 media for 30 min at 37°C and (C) A549 cells: Ad5T* or Ad5.HVR5* (2x10¹⁰ vp/mL) were incubated with RPMI-1640 media, 1–90% C57BL/6 mouse serum in RPMI-1640 media in the absence or presence of 10 μg/mL hFX for 30 min at 37°C. Virus suspensions were diluted 200-fold in serum-free media and 100 μL added to cells for 2 h at 37°C before being replaced with RPMI-1640 media with 2% FCS. Transgene expression was quantified ∼16 h post-transduction and relative light units (RLU) normalized to mg total protein. Results for each vector are shown as percentage of the matched media control. Groups that do not share the same letter (a, b, c, d) are significantly different to samples within that group. *p < 0.05 vs. matched controls.</p

FX-mediated Ad26.HVR5C cell surface binding and transduction.

<p>(A) Binding of 1000 or 5000 vp/cell of Ad5, Ad26 or Ad26.HVR5C was quantified after incubation with SKOV3 cells for 1 h at 4°C in the presence or absence of 10 μg/mL hFX. Vector genomes were detected by quantitative PCR. (B) SKOV3 cells were infected with 1000 or 5000 vp/cell of Ad5, Ad26 or Ad26.HVR5C in the absence or presence of 10 μg/mL hFX for 3 h at 37˚C, after which media was replaced by full RPMI-1640 media and cells incubated at 37˚C. Transgene expression was measured 48 h post-infection. *p < 0.001 vs. matched -/+FX controls.</p

Ad5 HVRs mediate neutralisation by naïve murine serum.

<p>SKOV3 (A) or A549 cells (B-C): Vectors (2x10¹⁰ vp/mL) were incubated with RPMI-1640 media, 90% C57BL/6 mouse serum in RPMI-1640 media in the absence or presence of 40 μg/mL Xbp for 30 min at 37°C. Virus suspensions were diluted 200-fold in serum-free media and 100 μL added to cells for 2 h at 37°C before being replaced with RPMI-1640 media with 2% FCS. Transgene expression was quantified ∼16 h post-transduction and relative light units (RLU) normalized to mg total protein. Results for each vector are shown as percentage of the matched media control. White bars = media alone, grey bars = serum and black bars = serum + Xbp. (D) C57BL/6 mouse serum was incubated with Ad (5x10¹⁰ vp/mL) in the presence or absence of 40 μg/mL Xbp for 90 min at 37°C. C3a levels were quantified by ELISA. *p < 0.005 vs. matched controls.</p

data_sheet_1_Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice.PDF

<p>Fibrocytes are myeloid lineage cells implicated in wound healing, repair, and fibrosis. We previously showed that fibrocytes are mobilized into the circulation after vascular injury, including the immune-mediated injury that occurs after allogeneic transplantation. A common response to inflammatory vascular injury is intimal hyperplasia (IH), which, alongside vascular remodeling, results in progressive loss of blood flow, downstream ischemia, and end-organ fibrosis. This forms the pathological basis of transplant arteriosclerosis and other diseases including post-angioplasty re-stenosis. In investigating whether fibrocytes contribute to IH, we previously showed that subpopulations expressing smooth muscle actin and CD31 are recruited to the site of injury and accumulate in the neointima. Expression of tissue factor (TF) by these “CD31+ myofibrocytes” is needed for progressive neointimal expansion, such that TF inhibition limits the neointima to a single layer of cells by day 28 post-injury. The aim of this study was to determine pathophysiological mediators downstream of TF that contribute to myofibrocyte-orchestrated IH. We first show that myofibrocytes make up a significant component of the neointima 28 days following injury. Using a previously defined adoptive transfer model, we then show that CD31+ myofibrocytes get recruited early to the site of injury; this model allows manipulations of the adoptively transferred cells to study how IH develops. Having confirmed that inhibition of TF on adoptively transferred cells prevents IH, we then show that TF, primarily through the generation of thrombin, induces secretion of angiopoietin-2 by myofibrocytes and this directly stimulates proliferation, inhibits apoptosis, and induces CXCL-12 production by neointimal cells, including non-fibrocytes, all of which promote progressive IH in vivo. Prior incubation to inhibit angiopoietin-2 secretion by or block TIE-2 signaling on adoptively transferred fibrocytes inhibits IH. These novel data indicate that angiopoietin-2 production by early recruited myofibrocytes critically influences the development of IH after vascular injury and suggest new therapeutic avenues for exploration.</p

<i>In vivo</i> analysis of Ad26.HVR5C biodistribution in immune deficient mice.

<p>(A + B) Luciferase expression was visualised (A) and quantitatively assessed (B) by whole-body bioluminescence (IVIS) imaging 72 h after intravascular administration of 1x10¹¹ vp/mouse Ad5, Ad26 or Ad26.HVR5C in the absence or presence of 133 μg/mouse warfarin (n = 4 animals/group) or PBS (n = 3 animals/group) in RAG^2-/- mice. (C) Livers were harvested 72 h post-injection and processed for quantification of luciferase transgene expression and (D) quantification of viral genomes, using 200 ng of DNA for analysis. *p<0.05 and NS (not significant) vs the matched non-warfarin treated control.</p

Generation of Ad26 hexon modified vectors and effects on FX binding by SPR.

<p>(A) Virus titers and vp/pfu ratios for each of the vectors produced. (B) SPR analysis of Ad5, Ad26 and mutant vector interaction with immobilized hFX (500 RU). Sensorgrams of 10¹¹ vp/ml of vector injected at a flow rate of 30 μL/min. (C i) SPR analysis of hFX for immobilized Ad26.HVR5C. Samples analysed in triplicate at 4 concentrations of hFX. (C ii) SPR analysis of hFX for immobilized Ad5. Samples analysed in triplicate at 7 concentrations of hFX. The black lines represent the fit to the data. (C iii) Kinetic constants of hFX for immobilized Ad5 or Ad26.HVR5C as determined by SPR.</p

<i>In vivo</i> analysis of Ad26.HVR5C mediated liver transduction in MF1 mice.

<p>(A + B) Luciferase expression was visualised (A) and quantitatively assessed (B) by whole-body bioluminescence imaging 72 h after intravascular administration of 1x10¹¹ vp/mouse Ad5, Ad26 or Ad26.HVR5C in the absence or presence of 133 μg/mouse warfarin (n = 6 animals/group) or PBS (n = 3 animals/group) in MF1 mice. (C) Livers were harvested 72 h post vector injection and processed for quantification of luciferase transgene expression and (D) quantification of viral genomes, using 200 ng of DNA for analysis. *p<0.05, ***p<0.001 and NS (not significant) vs the matched non-warfarin treated control.</p

FX protects Ad26.HVR5C against immune attack.

<p>(A) 2x10¹⁰ vp/mL of Ad5, Ad26 or Ad26 hexon modified vectors were incubated with RPMI-1640 media, 90% C57BL/6 mouse serum, 90% C57BL/6 mouse serum with 40 μg/mL X-bp, 90% RAG^2-/- mouse serum or 90% RAG^2-/- mouse serum with X-bp, for 30 min at 37°C. Virus suspensions were diluted 200-fold in serum-free media and 100 μL added to SKOV3 cells for 2 h at 37°C before being replaced with RPMI-1640 media with 2% FCS. Transgene expression was quantified ∼16 h post-transduction and relative light units (RLU) normalized to mg total protein. Results for each vector are shown as percentage of the matched media control. *p < 0.001 vs. matched media controls. (B) C57BL/6 mouse serum was incubated with Ad (5x10¹⁰ vp/mL) in the presence or absence of 40 μg/mL Xbp for 90 min at 37°C. C3a levels were quantified by ELISA. (C) C57BL/6 mouse serum was incubated with Ad5 or Ad5T* (5x10¹⁰ vp/mL) in the presence or absence of 20 μg/mL NapC2 for 90 min at 37°C. C3a levels were quantified by ELISA. *p < 0.005 vs. matched controls.</p