P53 Gene Therapy To Human Nasopharyngeal Carcinoma Cancer Using Recombinant Archaeal Histone-like Protein

With the deeper insight into the cancer immunity and mechanism, cancer gene therapy has greatly developed and become more and more rounded. The number of drugs relevant to gene therapy, which have been authorized to be used in clinic application, has increased annually. In order to transfer the gene into the target cell and express the relative protein successfully, vectors have been employed to realize the objective. Generally, there are two main types of gene delivery vectors: viral and non-viral. Viruses are the most effective gene delivery vehicles for gene therapy. However, their defects are obvious since they suffer from a number of undesirable properties for therapeutic applications, such as uncertainties about safety, immunogenicity, limited packaging capacity for genetic material and manufacturing difficulties. As an alternative approach, recent efforts have focused on developing non-viral gene delivery systems, which have the potential advantages as follows: ease of production and use, low toxicity and immunogenicity, and no limitation of the size or type of delivered nucleic acid. Among the non-viral vectors, there is a kind of protein which has the characteristic of DNA binding ability, and it can transfer DNA into the target cells to play the relative roles. Histone is considered to be one of the most attractive alternatives.In the present study, an archaeal histone-like protein HPhA from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain was employed as the carrier of naked plasmid DNA, which contained a wild type p53 tumor suppressor gene. The transfection efficiency and antitumor activity in human p53-deleted Nasopharyngeal Carcinoma cells (CNE) were evaluated in vitro and in vivo. Firstly, the ability of HPhA gene transfer into cells was evaluated as percentage of p53 staining positive cells 24 hours by standard immunohistochemical techniques, and the transfer efficiency of HPhA- transfected cells calculated through photomicrography was 75%, higher than other groups of cells. The results favored that HPhA could transfer the target gene into CNE cells efficiently. The p53 mRNA and p53 protein expression level were calculated by real-time PCR and Western blot analysis, and the results both indicated that the exogenous p53 gene of HPhA-transfected cells was translated into immunoreactive p53 protein sufficiently.Cell proliferation assay and flow cytometry detecting apoptosis were performed to investigate the influence of HPhA mediated p53 gene expression to cell cycle. The results showed the HPhA-mediated transfection of tumor suppressor gene p53 was effective in inducing apoptosis and inhibiting tumor cell growth. Compared with lipofectamineTM 2000, HPhA was more effective in mediating gene transfection. Encouraging results were also obtained in studies using tumor xenografted nude mice. Local HPhA/p53 treatment by intra-tumoral injection led to a significant inhibition of tumor growth, which was consistent with the in vitro effect of HPhA-p53 on human cancer cells.Furthermore, the immunogenicity of HphA was investigated in BALB/c mice, which provided a good base for safe and effective application of HphA. The mice were divided randomly, and then immunized by HphA, HphA combining with Freuds adjuvant and bovine serum albumin combining with Freuds adjuvant. Humoral immunoresponse was evaluated with the serum IgG level tested by ELISA, and the cellullar immunologic response was evaluated with stimulation indexes and levels of secreted IFN-γof splenocytes. It was shown that mice immunized with HphA produced no specific antibody, and the stimulation indexes and levels of IFN-γhad no difference from the control group. Therefore, HphA had no immunogenicity in mouse,and could not produce humoral and cellular immune response.In this thesis, HPhA proved to enhance the in vitro and in vivo efficiency of p53 gene transfer, and was a promising new strategy for p53 gene therapy. In all, HPhA might serve as a promising, wildly applicable and highly efficient tool for gene delivery and gene therapy.