Fig 1: Biochemical characterization of ADA payload. (a) Expression of mADA was evaluated by western blot analysis in supernatants and bulk (whole cell lysate) of non-infected (NI), R-LM113, THV_ADA, THV_ADA-SP, and infected SKOV3 cells three days after infection at MOI of 0.1 pfu/cell. (b) Adenosine deaminase activity was measured in the supernatants described in the panel (a). Statistical significance was calculated between non-infected cells (NI, black line) and all the samples (R-LM113, blue line; THV_ADA, orange line; THV_ADA-SP, green line) and is reported as * for p-value < 0.05 and ns for not statistically significant.
Fig 2: Generation of Adenosine Deaminase-armed targeted herpes viruses. (a) 40 N terminus aa of wild-type and SP-bearing murine ADA were analyzed by SignalP 5.0 server to evaluate the presence and proficiency of the signal peptide. Non-signal peptide (Other, yellow line) and Signal Peptide (SP, red line) moieties are reported as 0 (0%) to 1 (100%) probability. The cut site of SP (CS) is indicated as dashed green line. (b) Schematic representation of THVs expressing ADA and ADA-SP. The scFv targeting hHER2 (dark blue) is shown in glycoprotein D (light blue). (c) The yield of R-LM113, THV_ADA, and THV_ADA-SP oncolytic viruses was evaluated in SKOV3 cells infected at MOI of 0.1 pfu/cell. Yield was reported as pfu/mL obtained by plaque assay from two biological replicates of infection. (d) Virion infectivity was measured as gc/PFU ratio. Non-statistically significant differences were reported as Not Significant (ns).
Fig 3: Adult Gata1s mice have a defect in mature erythrocytes. (A) Sulfo-NHS-biotin was injected IV on day 0, and the fraction of biotinylated RBCs was quantified serially by streptavidin labeling and flow cytometry, N = 4. (B) Mice were treated with PHZ solution by intraperitoneal injection on days 0 and 1. Serial HCT of WT and Gata1s (G1s) mice are shown at different time points after induced acute anemia. Normalized recovery was calculated through dividing the HCT mean value at each time point by the mean value at day 0 before treatment. Mean ± SD are shown, N ≥ 3, age = 2 months. ∗∗∗∗P ≤ .0001 by 2-way ANOVA. (C-G) Determination of PB parameters associated with hemolytic anemia, including G6PD activity (C), reactive oxygen species (ROS) levels (D), CD55 and CD59a expression (E), lactate dehydrogenase (LDH) activity (F), and ADA activity (G). ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗∗P ≤ .0001 by the unpaired Student t test. MFI, mean fluorescence intensity; RFU, relative fluorescence units; PBRBC, peripheral blood red blood cell.
Supplier Page from Abcam for Adenosine Deaminase (ADA) Activity Assay Kit (Colorimetric)