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. 2022 Feb 28;28:58–76. doi: 10.1016/j.omtn.2022.02.019

Figure 3.

Figure 3

LVs encoding the Pol III-driven RNAi effectors produce potent VEGFA knockdown but also affect viability

(A) A schematic vector map of the pCCL-based lentiviral transfer vector encoding the U6-driven RNAi effectors. CMV, cytomegalovirus promoter; cPPT, central polypurine tract; EGFP, enhanced green fluorescent protein; ΔU3, LTR unique 3′ region (U3) with self-inactivating deletion; PGK, phosphoglycerate kinase 1 promoter; Ψ, packaging signal; R, LTR repeat region; RRE, Rev-responsive element; T6, T-rich Pol III termination signal; U5, LTR unique 5′ region; U6, U6 snRNA promoter; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (B and C) Knockdown efficacy of LVs encoding the U6-driven 12 and 13 agshRNA-shRNA pairs. HEK293 cells were transduced with the LVs encoding the RNAi effectors and then transfected with dedicated dual luciferase reporters. Rluc/Fluc ratio is the mean of triplicates ± SD normalized to agshS1. (D and E) Western blot-assessed VEGFA levels in 293-hVEGFA cells transduced with the LVs encoding the U6-driven RNAi effectors, quantification, and representative blot. The intracellular VEGFA levels are presented relative to the total protein content, while the extracellular samples are volume normalized. Mean of triplicates ± SD, normalized to NT control. (F) RT-qPCR quantification of the mRNA levels of VEGFA in 293-hVEGFA cells transduced with the LVs encoding the U6-driven RNAi effectors. Geometric mean of the PPIA-normalized VEGFA fold change (FC) with geometric SD relative to NT. (G) MTT-based cell number assessment of ARPE19 cells 5 days post transduction with the LVs encoding the U6-driven RNAi effectors. The background-subtracted absorbance at 570 nm relative to the untreated negative control (NC) is presented. Means of quintuplicates ± SD. The NT and NC are in triplicates. (G) A one-way ANOVA was performed followed by Šídák's multiple comparisons test between each of the agshRNAs and their corresponding shRNAs. The multiplicity-adjusted p values are reported. ∗∗∗∗p <0.0001.