Anti-tumor Activity Of Cationic Liposomes Combined With Adenovirus Containing Murine EBV-induced Molecule 1 Ligand Chemokine Gene

Anti-tumor Activity of Cationic Liposomes Combinedwith Adenovirus Containing Murine EBV-inducedMolecule 1 Ligand Chemokine GeneCell BiologyPh.D candidate: CAO Mei Supervisor: Prof. WEI YuquanEBV-induced moleculel ligand chemokine (ELC) , one of CC chemokines,chemoattracts DCs, T cells, B ceils, NK cells and progenior macrophages. ELC me-diates its effect through the specific G protein-coupled seven-transmembrane domainchemokine receptor CCR7. Adenoviral vector is one of the mostly used gene deliv-ery vectors for cancer therapy because of its high transfection efficiency. However,its application is limited due to .immune responses that reduce transgene expressionand decrease the efficacy of repeated administration. We constructed the recombi-nant replication deficient adenoviral vectors containing mELC gene (Ad-mELC) andcombined them with polyethylene glycol modified cationic liposome (PEG-liposome)for immunotherapy against murine malignant ascites.In AdEasy vector system, pAdEasy-1 is a 33.4 kb plasmid containing the ade-novirus serotype 5 (Ad5) genome with deletions of the E1 and E3 regions. Uponhomologous recombination in bacteria BJ5183 with a transfer vector pShuttle carry-ing the mELC gene cassette, the new plasmid pAd-mELC was generated with theexpression cassette inserted into the E1 region of the adenovirus genome. Subse- quently 293 cells were transfected with the plasmid pAd-mELC to generate a recom-binant adenovirus Ad-mELC expressing the desired gene product. The plaque assayand PCR analysis were performed to screen the replication deficient adenovirus, andour results demonstrated that no replication competent adenovirus (RCA) weredetected. Expression of mELC by Ad-mELC in vitro was examined by means ofRT-PCR and Western blot. The chemoattractant activity in lymphocytes was deter-mined by chemoattractant experiment, and its activity was markedly inhibited byanti-mELC monocloned antibody.On the other hand, the PEG -liposomes were synthesized. The average particlesize of PEG-liposome was 151nm, and zeta potential was 38.6mV, which demon-strated the PEG-liposome were relatively stable.The cytotoxicity was examined by MTr showing that the Ad-mELC was littletoxic on the tumor cells with MOI during the range from 50 to 800 (P＞0.1). Simila-rily treatment with PEG-liposome at the concentration of 25ng/10~3 cells displayedlittle toxicity (P＞0.1).With the establishment of the animal models for fibrosarcoma and hepatoma as-cites, the tumor-bearing mice were administrated by Ad-mELC combined withPEG-liposome (Ad-mELC/PEG) or Ad-mELC alone, and the controls were treatedwith, Ad-null, Ad-null/PEG, PEG-liposome or PBS, repectively. The average survivaltime of the Ad-mELC/PEG treated mice was 31.2d, which was 20 days, and 7.5 dayslonger than the average suvival time in PBS-treated, and Ad-mELC-treated mice, re-spectively.The ascites volume was measured 3 days after the second treatment. In the fi-brosarcoma model, the average ascites volume was 3.2 ml in Ad-mELC treated mice,and 3.5ml in Ad-mELC/PEG group, respectively, which are much lower than the av-erage ascites volume of 18.2ml in the PBS-treated group. Both the red cell and thetumor cell numbers in Ad-mELC and Ad-mELC/PEG treated.mice were significantlyreduced compared with the controls (P＜0.01). Similar results were observed in thehepatoma ascites models, as well as in the MethA fibrosarcoma ascites models. The mice were sacrificed, and the ascites were collected 15 days after tumorcells injection i.p.. Compared with the controls, the expression level of VEGF in theascites was much reduced in both Ad-mELC and Ad-mELC/PEG groups (P＜0.05).RT-PCR data showed that the expression of mELC was only detected in Ad-mELCand Ad-mELC/PEG groups.ELISA assay revealed that expression of mELC was down-regulated in ascitesof the Ad-mELC group 3 days after the final treatment. However, expression ofmELC in ascites kept at a high level in Ad-mELC/PEG group. Albeit no apparenttherapeutic differences was observed between Ad-mELC/PEG and Ad-mELC treatedmice after the second administration, the Ad-mELC/PEG treated mice can survivemuch longer than the Ad-mELC treated mice. The anti-tumor efficacy may be due, atleast partly, to the high level of mELC after the final administration and prolongedexpression of interferon-γ(INF-γ) and interleukin-12 (IL-12) in Ad-mELC/PEGtreated group examined by ELISA. The results suggest that PEG cationic liposomescombined adenovirus could prolong expression of the therapeutic gene in vivo andmay enhance the anti-tumor efficacy.In summary, Ad-mELC was generated, and subsequently was utilized for ma-lignant ascites gene therapy alone or in combination with PEG. The average survivaltime of the Ad-mELC/PEG treated mice was much longer than that in the controls.Our data suggested that prolonged expression of mELC in vivo with the aid ofPEG-liposome might enhance the anti-tumor activity. These data obtained in ourstudies underscore the clinical therapeutic potential of the combination of the recom-binant Ad-mELC with PEG-liposome mediated gene delivery system.