Figure 5.

In vivo bioactivity of AAVX-HPLC and iodixanol ultracentrifugation purified AAV

(A) Quantification of viral DNA and GFP RNA and protein levels in the liver, brain, and quadriceps of mice injected with a total of 10¹¹ vg/mouse of scAAV9-Cbh-GFP. N = 5 for both scAAV-GFP AAVX and scAAV-GFP iodixinal injected mice, and N = 2 for vehicle-injected mice. DNA and RNA were quantified using qPCR and qRT-PCR, respectively, and protein using Simple Wes. Statistical significance was assessed using two-way ANOVA with Šídák’s post hoc tests. Statistically non-significant differences are not shown on the figure, except for AAVX versus iodixanol groups. Note that the AAV DNA levels in the brain were likely below the limit of quantification in this assay. (B–D) Imaging analysis of livers sectioned, stained for tomato lectin and DAPI, and imaged for native GFP fluorescence, tomato lectin, and DAPI. (C) Comparison of native GFP averaged from 400–700 cells per animal. (D) Percentage of cells that are GFP positive, counted as cells with a higher mean fluorescence intensity than the highest mean fluorescence intensity observed in the vehicle group. Statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test for (C) and two-tailed t test for (D). ns, p > 0.05; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Error bars denote standard deviation in all panels.