B4GALNT2 Antibody NBP1-91229 from Novus Biologicals, a Bio-Techne Brand

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ACS nano
April 26 2022

Fig 1: In vivo dose optimization of B4galnt2 gene activation. (a) Images of liver sections showing RNAscope staining for B4galnt2 mRNA (green), Dolichos biflorus agglutinin (DBA) lectin staining (magenta), and DAPI (blue) at 1 day postinjection. Scale bar is 25 µm. (b) Flow cytometry plots showing DBA lectin staining of hepatocytes at 48 h using 1 mg/kg VPR mRNA and 1 mg/kg sgRNA. B4galnt2 mRNA copy numbers (c) and the percentage of activated hepatocytes (heps) (d) between formulation approaches at varying mRNA doses with constant sgRNA/mRNA mass ratio. Heat maps of B4galnt2 mRNA copies (e) and the percentage of activated hepatocytes (f) with varying mRNA and sgRNA amounts. (g) Direct measurement of LNP encapsulation of mRNA by qPCR. Data were normalized to the separate LNP formulation condition. Data are presented as mean ± SEM (n = 4 technical replicates). (h) VPR mRNA copy numbers from liver samples compared to theoretical doses. Linear regression was performed for each data set (solid line = best fit, dotted lines = 95% CI). (i) Overall delivery of VPR mRNA to livers for each formulation approach is reported as the slope of the linear fits from (h). Data were normalized to the separate LNP formulation condition. Data are presented as mean ±95% CI. (j) Dose-corrected qPCR results from combined and separate formulation experiments. (k) Dose-corrected flow cytometry results from combined and separate formulation experiments. Unless otherwise noted, data represent mean ± SEM (n = 3–4 mice). 4PL curves were fit to data in (d) and (k) (solid lines = best fit curve, dotted lines = 95% CI). An extra sum-of-squares F-test was performed to assess statistical significance between the EC50 values of 4PL fits and slopes of linear fits. When P > 0.05, a combined EC50 value was reported for the curves. Additional statistical significance was assessed using a two-way ANOVA followed by Dunnett’s multiple comparison (c,d,h) and a student’s t test (g,i) between formulation approaches (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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ACS nano
April 26 2022

Fig 2: In vitro gene activation of B4galnt2 in AML12 cells. (a) Single sgRNA screen by qPCR (top) and flow cytometry (bottom) at 24 h post-transfection. (b) Combinatorial screen of five sgRNAs by qPCR (top) and flow cytometry (bottom) at 24 h post-transfection. (c) Time course of gene activation by qPCR (left) and flow cytometry (right). (d) RNAscope assay against B4galnt2 mRNA (green) and DAPI (blue). Scale bar is 50 µm. B4 denotes B4galnt2 sgRNAs. NT denotes nontargeted sgRNAs. UT denotes untreated mice. RQ (relative quantification) denotes the fold change of B4galnt2 mRNA in treated samples relative to untreated samples and normalized to Gapdh mRNA for both. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was assessed using a two-way ANOVA followed by Dunnett’s multiple comparison between the nontargeted condition (****P < 0.0001).
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ACS nano
April 26 2022

Fig 3: In vivo demonstration of optimized B4galnt2 gene activation. (a) Representative slide scan images of liver sections showing RNAscope staining for B4galnt2 mRNA (green) and DAPI (blue). Insets depict the relative locations of 4× and 16× views. Scale bars are 800 µm for 1×, 200 µm for 4×, and 50 µm for 16× images. Time courses of B4galnt2 mRNA (b) and activator mRNA (c) copy numbers from liver tissue over 9 days. B4galnt2 mRNA copy numbers (d) and percentage of activated hepatocytes (e) in mice treated with activator mRNA and B4 sgRNA with or without AcrIIA4 co-delivery. The “–AcrIIA4” groups were dosed with activator mRNA and B4 sgRNA on day 0. The “+AcrIIA4” groups were simultaneously dosed with activator mRNA, B4 sgRNA, and AcrIIA4 mRNA on day 0. All VPR and VPH-SS18-treated mice were euthanized at day 1 postinjection, and all p300-treated mice were euthanized at 5 days postinjection. (f) VPH-SS18 time course with redosing. VPH-SS18 mRNA and B4 sgRNAs were delivered to both groups on day 0. AcrIIA4 treatment was given to one group on day 5, followed by euthanasia on day 6. Remaining mice that did not receive AcrIIA4 mRNA were then redosed with VPH-SS18 mRNA and B4 sgRNAs on day 14. Data represent mean ± SEM (n = 3–4 mice). Statistical significance was assessed using a two-way ANOVA followed by Dunnett’s multiple comparison compared to the NT-treated group (b,c) and a student’s t test (d,e) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).