Experimental Human Liver Cancer-specific Dual Gene Therapy With Pegylated Immuno-lipopolyplexes (PILP)

ChapterⅠ:Pegylated immuno-lipopolyplexes:A novel non-viral gene delivery system for liver cancer therapyObjective:To develope an efficient and safe non-viral delivery system that delivers the therapeutic genes to hepatocellular carcinoma in vivo for liver cancer gene therapy.Methods:Pegylated immuno-lipopolyplexes (PILP), a novel and efficient non-viral gene delivery system was developed by employing DNA/polyethylenimine (PEI 25 kDa) polyplexes, as well as anionic liposomes composed of POPC, (DSPE)-PEG2000 and (DSPE)-PEG2000-biotin, at five different lipid/DNA molar ratios (50/1,90/1,130/1,170/1 and 210/1), and by using streptavidin-monoclonal antibody conjugating through the biotin group located at the distal end of the PEG spacer as targeting antibody.Results:This vector was highly effective in protecting DNA from enzyme digestion, and stable in particle size and zeta potential even after 20 day-storage at 4 ℃. At the lipid/DNA molar ratio 170/1, the PILP were found to have the highest in vitro transfection efficiency with an average particle size of 132 nm and an average zeta potential of +9.5 mV. These complexes showed high efficiency in gene delivery to liver cancer cells with no significant cytotoxicity. Interestingly, the in vitro transfection efficiency did not decrease significantly up to 10 days of storage of PILP at 4℃. Intravenous administration of the PILP resulted in tumor and liver targeted gene expression of the reporter genes enhanced green fluorescent protein (EGFP) and luciferase as opposed to the lung targeted gene expression obtained with PEI/DNA complexes, causing no cytokine production and liver injury.Conclusions:We conclude that the PILP are promising gene delivery systems which may be used to target the liver cancer.Chapter II:Combined RNA interference gene therapy targeted at hEGFR mRNA and hTERT mRNAObjective:To establish the feasibility and efficacy of combination nonviral hEGFR and hTERT gene therapy for experimental human liver cancer with the pegylated immuno-lipopolyplexes (PILP).Methods:Two expression plasmids encoding a short hairpin RNA directed at nucleotides 2627-2655 with the human EGFR mRNA and directed at nucleotides 1031-1059 with the human TERT mRNA (anti-hEGFR pDNA and anti-hTERT pDNA) were formulated as PILP respectively with receptor-specific monoclonal antibodie (MAb), the murine 83-14 MAb to the human insulin receptor. Human hepatoblastoma SMMC-7721 cells were transfected with these PILP. TR-PCR was used to detect the silencing of the hEGFR mRAN and hTERT mRAN on 4 day after transfection. Cell cycle distribution and apoptotic rate were evaluated by flow cytometry (FCM) on 2,4,6 day after transfection. SMMC-7721 cells were implanted subcutaneouly into scid mice, and every 5 day i.v. gene therapy was started at day 10 after implantation of 2000,000 cells. To calculate the tumor growth inhibition rate and survival time of mice. To detect the hEGFR protein expression in tumor tissue with Immunohistochemistry and Western Blot and detect the downregulation of the hEGFR mRNA by RT-PCR.Results:a short hairpin RNA inhibited human EGFR (hEGFR) gene expression in a time-dependent manner in cultured hepatoblastoma cells. In SMMC-7721 cells treated with anti-hEGFR pDNA and con-treated with anti-hEGFR pDNA and anti-TERT pDNA, these plasmid all formulated as PILP with 8314 MAb, we displayed sequence specific silencing of hEGFR with 72.53% and 78.68% respectively; the percentage of cells in G0/G1 phase were increased by 17.62% and 45.62% respectively compared with controls; the total apoptotic rate were 12.47% and 29.59% respectively. In two groups i.v. anti-hEGFR pDNA and con-i.v. anti-hEGFR pDNA and anti-hTERT pDNA, these plasmid all formulated as PILP with 8314 MAb, every five days i.v. RNAi gene therapy resulted in 66.71% and 89.01% suppression of hEGFR mRNA,56.72% and 80.73% of down-regulation of EGR protein production,46.13% and 74.04% of tumor gowth inhibition rates, 76.47% and 94.12% increase in survival time of mice with liver cancer.Conclution:Combined hEGFR and hTERT gene therapy could caused more significant suppression for human hepatoblastoma SMMC-7721 cells in vivo and vitro compare one gene therapy. Combination of the two gene therapy may be additive or synergistic in antitumor effect.ChapterⅢLiver-specific RNA interference gene therapy for experimental human liver cancerObjective:To minimize the possible adverse effects caused by non-target gene expression by using liver-specific promoter and pegylated immuno-lipopolyplexes (PILP) technique.Methods:The CMV promoter of the enhanced green fluorescent protein (EGFP) expressing plasmid CMV-EGFP and the U6 promoter of the expression plasmid encoding a short hairpin RNA directed at nucleotides 1031-1059 within the human TERT mRNA (pU6-shTERT) were replaced with the liver-specific promoter apolipoprotein A-I (ApoAI) generating ApoAI-EGFP plasmid and ApoAI- shTERT plasmid. Cell lines HepG2, SMMC-7721, Lo2, MCF7, TF-1a, ACC-2 and IMR90 were transfected by pApoAI-EGFP, pCMV-EGFP, pApoAI-shTERT, pU6-shEGFP and pU6-shTERT with ExGen 500 in vitro transfection reagent. To qualitatively visualize expression of EGFP with fluorescent microscope on 48 h after Transfection and detect telomerase activity on 72 h after transfection. Either pCMV-EGFP or pApoAI-EGFP was formulated as PILP, a novel and efficient gene delivery system, with receptor-specific monoclonal antibodie (MAb), the rat 8D3 MAb to the mouse transferring receptor. H22 tumor-bearing mice were intravenously administered with 40μg/mouse of the PILP. Either pApoAI-shTERT or pU6-shEGFP was formulated as PILP with MAb, the murine 83-14 MAb to the human insulin receptor. Human hepatocellular carcinoma SMMC-7721 cells were implanted subcutaneouly into scid mice, and every 5 day i.v. gene therapy was started at day 10 after implantation of 2000,000 cells.Results:In vitro expression experiments performed in various cell lines including HepG2, SMMC-7721, MCF-7, ACC-2 and Lo2 indicated that pCMV-EGFP treatment caused gene expression in all the cell lines, whereas pApoAI-EGFP treatment only induced EGFP expression in cells of liver origin including the liver cancer cells HepG2 and SMMC-7721 and the normal liver cells Lo2; pU6-shTERT treatment inhibited the growth and telomerase activity in all the hTERT-expressing cancer cells, whereas pApoAI-shTERT treatment only inhibited the growth and telomerase activity in hTERT-expressing cancer cells of liver origin only including the liver cancer cells HepG2 and SMMC-7721. In vivo expression experiments performed in H22 tumor-bearing mice indicated that there was significant EGFP expression in liver, tumor, spleen, brain and lung in the pCMV-EGFP treated mice, whereas in the ApoAI-EGFP treated mice there was only gene expression in liver and tumor and the non-liver organ gene expression was eliminated. RNA interference gene therapy performed in SMMC-7721 tumor-bearing mice treated with pApoAI-shTERT caused 22.26% the tumor growth inhibition rate and an 50% increase in survival time of mice.Conclusions:The use of the PILP technology and liver-specific promoter enables efficient and liver-specific expression of an exogenous gene and enables liver-specific RNA interference gene therapy.