Zhenzhong Zhang

Pegylated immuno-lipopolyplexes: A novel non-viral gene delivery system for liver cancer therapy

In this study, pegylated immuno-lipopolyplexes (PILP), a novel and efficient 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. 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 degrees C. 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 degrees C. Intravenous administration of the PILP resulted in tumor and liver targeted gene expression of the reporter genes EGFP and luciferase as opposed to the lung targeted gene expression obtained with PEI/DNA complexes, causing no cytokine production and liver injury. We conclude that the PILP are promising gene delivery systems which may be used to target the liver cancer.

Combinational RNAi gene therapy of hepatocellular carcinoma by targeting human EGFR and TERT

Both human telomerase reverse transcriptase (hTERT) and epidermal growth factor receptor (hEGFR) are ideal targets for RNA interference (RNAi)-based gene therapy of hepatocellular carcinoma. Two shRNA expression plasmids pU6-shTERT and pU6-shEGFR targeting hTERT and hEGFR, respectively, were separately formulated as pegylated immuno-lipopolyplexes, a novel non-viral gene delivery system. In vitro studies showed that when pU6-shTERT and pU6-shEGFR were combined and applied to SMMC-7721 cells, there was a significant additive effect on cytotoxicity as well as cell apoptosis, compared to pU6-shTERT or pU6-shEGFR alone, with a cell viability of 50.9±7.4%, 79.2±3.6% and 77.1±3.6%, respectively, and with a cell apoptotic rate of 44.8±0.9%, 25.1±0.4% and 29.5±0.8%, respectively. In vivo study in SMMC-7721 xenograft tumor model demonstrated that intravenous administration of PILP-formulated pU6-shTERT and pU6-shEGFR caused an additive effect on tumor growth inhibition, compared to pU6-shTERT or pU6-shEGFR alone, with a tumor growth inhibition rate of 74.0%, 36.3% and 46.1%, respectively, which is consistent with the downregulated EGFR and TERT mRNA expression. The results suggest that combinational RNAi gene therapy of hepatocellular carcinoma by targeting human EGFR and TERT with pegylated immuno-lipopolyplexes is a new and good strategy.

Liver-specific gene therapy of hepatocellular carcinoma by targeting human telomerase reverse transcriptase with pegylated immuno-lipopolyplexes.

The purpose of this study is to explore the possibility and feasibility of liver-specific gene therapy. A shRNA expression plasmid against human telomerase reverse transcriptase (hTERT) was constructed under the control of liver-specific promoter apolipoprotein A-I (ApoAI), designated as pApoAI-shTERT, and its liver-specific cytotoxicity and inhibition of telomerase activity were first evaluated in different cell lines, and its therapeutic effect was further studied in SMMC-7721 human liver tumor-bearing mice in vivo. The results showed that compared to pU6-shTERT, a shRNA expression plasmid against hTERT under the control of U6 promoter, pApoAI-shTERT only significantly diminished the cell viability in the telomerase positive hepatocarcinoma cells and showed no cytotoxicity in the telomerase negative cell lines as well as in the telomerase positive cell line of non-liver origin. Besides, pApoAI-shTERT only significantly reduced telomerase activity in the telomerase positive cell lines of liver origin. Intravenous administration of pegylated immuno-lipopolyplexes (PILP) formulated green fluorescent protein (GFP) expression plasmid under the control of ApoAI into liver tumor-bearing mice resulted in restricted GFP expression in liver and liver tumor. The treatment of pApoAI-shTERT formulated as PILP caused a 56% increase in the life span of SMMC-7721 tumor-bearing mice in vivo relative to the control, which was in agreement with the reduced tumor size and down-regulated hTERT mRNA level in the tumors. We conclude that the vector pApoAI-shTERT was able to cause liver-specific and hTERT target-specific cytotoxicity, and utilizing PILP to deliver pApoAI-shTERT is a promising strategy for liver-specific gene therapy.

Liver-specific expression of an exogenous gene controlled by human apolipoprotein A-I promoter

Liver-specific gene therapy is advantageous to minimize the possible adverse effects caused by non-target gene expression. The CMV promoter of the enhanced green fluorescent protein (EGFP) expressing plasmid pCMV-EGFP was replaced with the liver-specific promoter apolipoprotein A-I (ApoAI) generating pApoAI-EGFP plasmid. In vitro expression experiments performed in various cell lines including HepG2, SMMC-7721, MCF7, 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. Either pCMV-EGFP or pApoAI-EGFP was formulated as pegylated immuno-lipopolyplexes (PILP), a novel and efficient gene delivery system. Following intravenous administration of the PILP in H22 tumor-bearing mice, there was significant EGFP expression in liver, tumor, spleen, brain and lung in the pCMV-EGFP treated mice, whereas in the pApoAI-EGFP treated mice there was only gene expression in liver and tumor and the non-liver organ gene expression was eliminated. This study suggests that the use of the PILP technology and liver-specific promoter enables efficient and liver-specific expression of an exogenous gene.