| Part ⅠIn the recent ten odd years, adenovirus (Adv) has been one of the effective vectors for gene therapy. Up to now, more than 1000 therapy protocols have been approved in the world, 26% of which used Adv to deliver therapeutic genes. To meet the increasing needs of Adv for pre-clinic and clinic gene therapy programs, researchers are badly in need of developing an Adv production technology of efficient, large-scalable producing and reproducible process in order to make a great lot of adenoviral gene drugs up to clinic standards. Perfusion culture of 293 cells is one of the most commonly used methods in producing adenovirus vectors and it is specially suitable for industrialized production. We have carried out experimental studies about producing adenovirus type 5 containing the GFP gene under the control of the CMV promoter with perfusion culture of 293 cells. The results showed that the suitable perfusion rate was 1.5-2 volume/day and the best time of collecting cells was 48 hours after infection by Ad-GFP. In this way, the cell-specific productivity was around 18200±1700VP/cell. The results also showed that the cell-specific and volumetric productivity of Ad-GFP were higher than other situations when the 293 cells were infected at the density of 3.0><106 cells/ml. According to the experimental data, we have developed the process of producing Ad-GFP using perfusion culture of 293N3S cells in a 5L bioreactor and produced a great quantity of high titered Ad-GFP.Part Ⅱ The traditional purification method of recombinatant adenovirus vectors was CsCl gradient centrifugation. This method can only produce a small amount of adenovirus and the most important thing is that the method can not be effectively enlarged to be suitablefor industrialized production. In the last few years, the anion-exchange chromatography has become the common way for purifying adenovirus vector for it can give better results. The continuous two-step anion-exchange chromatography was applied to purifying Ad-GFP in our experiments. The Q Sepharose XL was used in the first step of purification, and the effluent was purified by the second sort anion-exchange resin, Source 15Q. Our results demonstrated that some hybridproteins were removed from the cell lysate by the Q Sepharose XL, but we could not get purer adenovirus without the second step of purification using Source 15Q. The final recovery of Ad-GFP was about 50 per cent through two anion-exchange chromatographies, and the Ad-GFP was confirmed by HPLC and SDS-PAGE. The Ad-GFP coming from the chromatography was similar to that from CsCl gradient centrifugation. This time- and labor-saving process could be flexibly enlarged and is suitable to large-scale production.Part HI p53 gene mutation is present in most human tumors and is one of the important pathogenic mechanisms in cancer development. It was reported that p53 gene mutation was found in more than 75 per cent of lung cancer and was closely correlated with the carcinogenesis and growth of lung cancer. The mutated p53 gene not only promotes cancer cell growth and proliferation, but also affects the genomic stability. In consequence, it accelerates the canceration of cells. Wild-type p53 gene has become one of the most commonly used anti-oncogene and it plays an anti-cancer role through many pathways. So the wild-type p53 gene has been one of the main delivering gene in gene therapy of cancers at present. GM-CSF, a cytokine, plays an important role in maturing and demonstratingfunctions of the antigen presenting cells. The exogenous GM-CSF gene was imported into tumor cells and continuously expressed in local tissue to form regional microenvironment with high concentration of GM-CSF. For antigen presenting cells, the microenvironment can promote the maturation and capability of presenting and processing tumor antigen. B7.1 is an immune co-stimulatory molecule belong to the immunoglobulin superfamily and can activate T cells together with second messenger to take part in cell immunity. We tansduced the co-stimulatory molecule gene into tumor cells without the second messenger, resulting in its effective expression. By doing so T cells can be induced to form a powerful anti-tumor immune reaction. The BB102 anticancer agent is the adenovirus, constructed by our laboratory, carrying wild-type p53 gene and tumor immunity related genes GM-CSF, B7.1. Our experiments were carried out to observe the therapeutic effect on Lewis lung carcinoma in C57 mice. It was shown that medium dose of BB102 had significant therapeutic effect on Lewis lung cancer. In addition, when combined with chemotherapy or radiotherapy BB102 remarkably increased chemotherapy or radiotherapy sensitivity of cancer cells. There are clinical reports showing the marketed recombinatant adenovirus-p53 agent can increase the radiosensitivity of cancer cells but there is no report demonstrating these agents could increase the chemosensitivity of cancer cells.
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