Figure 4

The expression of self-complementary AAV2-TTR-RB15 (scAAV2-TTR-RB15) vector in HepG2 cells. (A). The drawings depict the predicted conformations of monomer and dimer of TTR-RB15 virions. (B). Alkaline agarose gel electrophoresis (0.8%) of scAAV2-TTR-RB15 viral DNA, followed by Southern blot analysis, hybridized with ³²P-labeled RB15 probe. The positions of monomer and dimmer are shown. (C). Comparison of the ribozyme RB15 RNA levels between cells treated with non-modified parent rAAV2-TTR-RB15 vectors and cells treated with scAAV2-TTR-RB15 vectors at days 1 and 3 after treatment. Total RNA was extracted from HepG2 cells treated with either non-modified parent AAV2-TTR-RB15 or scAAV2-TTR-RB15 vectors. The levels of RB15 RNA were determined using one-step RT-PCR and analyzed by 2% agarose gel electrophoresis. The same samples were also determined by PCR only. The position of RB15 RNA is shown. (D). Comparison of apoB mRNA levels in HepG2 cells at day-3 after treatment with non-modified parent rAAV2-TTR-RB15 vectors or with modified scAAV2-TTR-RB15 vectors. ApoB mRNA was determined by real-time quantitative RT-PCR. The apoB mRNA levels were normalized with 18S RNA. The results are expressed as % of cells treated with inactive RB15-mutant. The results are shown as means ± standard deviation of triple experiments. Experiment of cells infected with adenovirus-RB15-mutant, adenovirus-RB15 (positive control) are performed at the same time and used as a positive comparison purpose. Cells treated with parent-rAAV2-TTR-RB15-mutant, parent rAAV2-TTR-RB15, scAAV2-TTR-RB15-mutant, and scAAV2-TTR-RB15 are shown. The p values analyzed by student t-test are shown. ns = not significant