Fig. 1: Improvement and optimization of the viral backbone. | Nature Communications

Fig. 1: Improvement and optimization of the viral backbone.

From: The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders

Fig. 1

a Sp1 and NF-kB binding sites were introduced upstream from EFS-NC promoter (pEFS-NC) (construct 1). The backbone integrated with 2xSp1 (two yellow circles) is outlined in construct 2. 4xSp1 (four yellow circles) is highlighted in construct 3. The backbone integrated with 2xNF-kB (two red circles) inserted upstream from pEFS-NC is outlined in construct 4. The backbone carrying 4xNF-kB (four red circles) is highlighted in construct 5. 2xSp1/2xNF-kB is depicted in construct 6. The complete EF1a promoter-driven plasmid is outlined in construct 7. The vector carried eGFP – nano-Luciferase reporter. b Physical titer of the modified AAV vectors. The lane order is the same as in the legend to (c). The experiment was done in triplicates. The statistical analysis was done using Prism GraphPad software. Ordinary one-way ANOVA was carried out to determine significant difference in the samples’ means ± SEM (P = 0.0002). ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01. ***P ≤ 0.001, ****P ≤ 0.0001; multiple comparisons of means were determined using Tukey’s multiple comparisons test (c) The expression of the modified AAV vectors has been assessed in HEK293T cells. The Luciferase assay was used to determine Relative Light Units (RLU) signals. Lane 1: EFS-NC core promoter- harboring vector packaged into AAV2.9 particles. Lane 2: EFS-NC core promoter- harboring 2xSp1 vector packaged into AAV2.9 particles. Lane 3: EFS-NC core promoter- harboring 4xSp1vector packaged into AAV2.9 particles. Lane 4: EFS-NC core promoter- harboring 2xNF-kB vector packaged into AAV2.9 particles. Lane 5: EFS-NC core promoter- harboring 4xNF-kB vector packaged into AAV2.9 particles. Lane 6: EFS-NC core promoter- harboring 2xSp1 and 2xNF-kB vector packaged into AAV2.9 particles. Lane 7: EF1-alpha complete promoter vector packaged into AAV2.9 particles. The experiment was done in quadruplicates. The statistical analysis was done using Prism GraphPad software. Ordinary one-way ANOVA was carried out to determine significant difference in the samples’ means ± SEM (P < 0.0001). ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01. ***P ≤ 0.001, ****P ≤ 0.0001; multiple comparisons of means were determined using Tukey’s multiple comparisons test.

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