Pseudomonas exotoxin antisense RNA selectively kills hepatitis B virus infected cells

Figure 1 A: p92 has the promoter and stem loop sequence of the T7 phage gene 10 and the Fd phage transcription terminator. In p95, p100 and p77, the LacZ-gene NotI-fragment of p92 is replaced by fragments containing truncated mutated forms of Pseudomonas exotoxin (PE38). p92 represents the control β-Gal expressing plasmid. p95 contains the gene for PE38 in reverse orientation to the CMV promoter. p77 expresses a non-functional frameshift mutant of PE38. p100 expresses the fully active PE38. B: Reverse transcription of toxin antisense RNA in vitro. Ethidium bromide stained nondenaturing 2% agarose gel. PCR fragments indicate the presence of toxin cDNA; templates were p100 RNA incubated with (1) no M-MLV RT, no random hexamer primers, (2) no M-MLV RT, random hexamer primers, (3) M-MLV RT, no random hexamer primers, (4) M-MLV RT, random hexamer primers.

Figure 2 A: Cytotoxicity of toxin expression plasmids to HepG2 and HepG2. 2.15 cells. β-Gal activities obtained after cotransfection with the β-Gal expressing DNA plasmid p92 (7.5 &mgr;g) and p92 plasmid without insert (7.5 &mgr;g) were set to 100%. Activities resulting from coexpression of toxin plasmids (p77, p95, p100) and β-Gal expressing plasmid p92 are expressed as relative levels. p95, coexpression of p95 with the toxin gene in opposite orientation to the CMV promoter; p100, coexpression of p100 with the active toxin fragment under control of the CMV promoter. Cell viability after cotransfection was determined by β-Gal activity from the β-Gal expressing plasmid. Reduced activity reflects cytotoxicity. B: Cytotoxicity of toxin of sense and antisense RNA. Transfection of PE sense and antisense RNA in HepG2 and HepG2.2.15 cells. RT dependent toxicity of antisense toxin RNA in HepG2 and HepG2.2.15 cells was determined. The β-Gal activities obtained after cotransfection with the β-Gal expressing DNA plasmid p92 (7.5 &mgr;g) and in vitro transcribed r92 control RNA (7.5 &mgr;g) were set to 100%. Activities resulting from coexpression of toxin RNA (r77, r95, r100) and β-Gal expressing plasmid p92 are expressed as relative levels. r77, antisense RNA of inactivated toxin from p77; r95, sense toxin RNA from p95; r100, antisense RNA of active toxin from p100.

Figure 3 RT-PCR of genomic DNA from HepG2 cells transfected with a HBV polymerase expression plasmid. 7.5 &mgr;g p77 RNA mixed with 7.5 &mgr;g p92 β-Gal expression plasmid were transfected in each experiment as well as plasmids expressing either wild-type or mutant HBV polymerase (7.5 &mgr;g each). Genomic DNA was extracted and analyzed by non-denaturing 2% agarose gel electrophoresis. PCR fragments indicate the presence of toxin p77 cDNA. A: Bio-Rad marker 50-2000 base pairs; B: PCR amplified fragment of HepG2 cells cotransfected with pCH391wt and r100; C: PCR amplified fragment of HepG2 cells cotransfected with pCH391mt and r100; D: P77 DNA as control DNA.

Figure 4 Cytotoxicity of toxin sense and antisense RNA to DHBV infected duck hepatocytes in the presence or absence of of 10 mmol/L adefovir. 7.5 &mgr;g RNA mixed with 7.5 &mgr;g p92 β-Gal expression plasmid were transfected in each experiment and β-Gal activities were quantified as described in legend to Figure 5. In addition, a wild-type HBV polymerase expression plasmid was transfected (7.5 &mgr;g). P92, control RNA from p92; p77, antisense RNA of inactivated toxin from p77; p95, sense toxin RNA from p95; p100, antisense RNA of active toxin from p100.

Figure 5 A: Cytotoxicity of toxin sense and antisense RNA to HepG2 cells in the presence or absence of transfected HBV polymerase expression plasmid. The β-Gal activities obtained after cotransfection with the β-Gal expressing DNA plasmid p92 (7.5 &mgr;g), in vitro transcribed r92 control RNA (7.5 &mgr;g) and plasmids expressing either HBV harbouring a wild-type or mutant HBV polymerase (7.5 &mgr;g each) were set to 100%. Values resulting from coexpression of toxin RNAs (r77, r95, r100) with p92 and either wild-type HBV polymerase or mutant HBV polymerase expressing plasmids are expressed as relative levels. B: Cytotoxicity of toxin sense and antisense RNA to HepG2 cells in the presence or absence of 10 mmol/L adefovir. 7.5 &mgr;g RNA mixed with 7.5 &mgr;g p92 β-Gal expression plasmid were transfected in each experiment and β-Gal activities were quantified as described in legend to Figure 3. In addition, a wild-type HBV polymerase expression plasmid was transfected (7.5 &mgr;g). p92, control RNA from p92; p77, antisense RNA of inactivated toxin from p77; p95, sense toxin RNA from p95; p100, antisense RNA of active toxin from p100.