P53 Gene Therapy To Human Nasopharyngeal Carcinoma Cancer Using Histone From Hyperthermophilic Archaeon Pyrococcus Horikoshii OT3

Gene therapy is becoming an important strategy in the treatment of various diseases such as cancer,AIDS,cystic fibrosis and vaccination. Effective gene transfer is an important prerequisite for gene therapy. The development of novel widely application DNA transfection system is an important goal of current molecular biological research. Generally, there are two main types of gene delivery vector: viral and non-viral. Viral vectors, the current favorites for intracellular DNA delivery, suffer from immunogenicity,nonspecificity and inherent risks of complications such as interference with the activity of tissue-specific promoters. Non-viral delivery systems are expected to have little or no harmful side-effects. The development of such systems with high efficiency,avoiding toxic and immunogenic problems, is essential and urgent. One approach focuses on the use of DNA-binding proteins as carriers of foreign genes into the host cell. The potential advantages of protein gene transfer include ease of use, production, purity, homogeneity, ability to target nucleic acids to specific cell types, the potential for cost-effective large-scale manufacture and the lack of limitation on production costs.We have previously described an archaeal histone-like protein-based (HPhA) gene delivery system and showed that HPhA formed stable non-covalent complexes with nucleic acids and improved their delivery by usingβ-galactosidase as a reporter gene. HPhA, an archaeal histone-like protein shares the properties of eukaryal histones: small, basic, toroidally wrap DNA, protect DNA from nuclease digestion and/or form nucleosome-like structures. These features make it potential carrier of gene deliveries.The p53 gene has been extensively studied and is known to play a critical role in cell regulation and control of apoptosis. Gene therapy using the p53 gene has been proposed for and performed with inactivation of p53 function. However, efficient and safe gene delivery remains a key issue for p53 based gene therapy. Because the functional loss of p53 gene was now well recognized as the most common event in carcinognesis, it was not surprising that wild-type p53 gene introduction had recently been used as a cancer gene therapy.In the present study, we attempted to use HPhA with naked plasmid DNA, which contains a wt-p53 tumor suppressor gene, and we evaluated the transfection efficiency and antitumor activity in human p53-deleted Nasopharyngeal Carcinoma (CNE) cells in vitro and in vivo. We observed that p53 delivery resulted in a more differential growth inhibition pattern in cancer cells in vitro and in vivo. The results showed a significant efficiency of gene transfer and gene expression. Higher transfection of HPhA/p53 complexes in CNE cells seemed to be responsible for higher expression of p53 mRNA. Efficient cell growth inhibition by HPhA/p53 complexes could be due to the increase of the expression of p53 proteins as well as due to the effect of histones on the proliferation of CNE cells. When using histones as DNA vectors, excess of the added histones could potentially interfere with cellular histones, and play regulatory function in transcription and slowed the cell cycle progression. Flow cytometric analysis suggested that the cytotoxic effect of HPhA/p53 was the result of apoptosis and cell cycle arrest. 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 consistent with the in vitro effect of HPhA-p53 on human cancer cells.Thus, it was supposed that functional exogenous p53 could be efficiently delivered in cancer cell lines using HPhA as a nonviral vector. Serum did not exert any inhibition of the transfection mediated by HPhA, the HPhA/p53 complexes could improve the stability of plasmid DNA in the presence of serum. In contrast, many cationic lipid-based transfection systems were inactivated by medium containing as low as 5-10% serum. Thus, the higher transfetion efficiency of plasmid DNA may be attributed to the enhanced stability of plasmid HPhA/DNA complexes. Moreover, the enhanced mRNA expression of p53 may increase the protein levels of p53 in the cells transfected with HPhA/p53 complexes compared to the cells treated with Lipofectamine complexes or the plasmid alone. And the penetration of histone into tissue-cultured cells occurring by direct translocation through the cell plasma membrane and not by a typical endocytosis, may facilitate the escape of DNA from the endosome. HPhA may be highly efficient gene delivery.Efficient gene delivery is one of the most important tasks in current non-viral gene therapy research. An ideal vector may find its application for in vivo and in vitro transfection of cells or grafts for transplantation and for various protocols of a localized gene therapy. Nuclear proteins, such as histones mediated gene transfer system might be an ideal system for gene transfer. Histones, which were natural DNA binding and condensing proteins, offered good advantages to serve as a safe and efficient gene delivery tool. Histones had been demonstrated to have higher efficiency of gene transfer compared with other non-viral transfer systems. Therefore, it was thought to be one of the most attractive alternative vectors.This study was designed to evaluate the efficiency and potential applicability of HPhA carrier system mediated p53 gene therapy. The results showed the HPhA-mediated transfection of tumor suppressor gene p53 was effective in inducing apoptosis and inhibiting tumor growth. Our results suggested the HPhA-mediated p53 delivery might have the potential for clinical application of nonviral vector mediated cancer therapy.The fate of gene delivery vectors in the body, i.e., their pharmacokinetics, is also another important factor in in vivo gene therapy. The pharmacokinetics determines the availability of the vector following in vivo administration. To our knowledge, no literature information is available on the pharmacokinetics of HPhA or other related histone.A competitive direct enzyme-linked immunosorbent assay (ELISA) has been developed to determine concentrations of an archaeal histone-like protein (HPhA) in mice plasma. Direct ELISA can be regarded as the simplest form of ELISA, of which there are two formats. One is antibody-coated ELISA, and the other is antigen-coated ELISA. In this study, the first format was developed to determine HPhA concentrations in rat plasma used for the investigation of the pharmacokinetics of HPhA. The most widely used enzymes for labeling are horseradish peroxidase (HRP), alkaline phosphatase (AP), andβ-galactosidase. In this study, horseradish peroxidase was selected to label gossypol because HRP had been proved with high turnover number and stability.Firstly, Anti-HPhA monoclonal antibody was purified by ammonium sulfate precipitation and ProteinA AffinityPak. The antigen (HPhA) was labeled with horseradish peroxidase by periodate oxidation method. Both the purified antibody and the enzyme-labeled HPhA were employed to develop cdELISA to facilitate further analysis of HPhA pharmacokinetics. The purified antibody was diluted in a coating buffer, carbonate/bicarbonate buffer (pH 9.6). After coating, any excess free antibody was removed by a washing step with a neutral buffered solution (PBST). The blocking solution (1% BSA in PBS, containing 0.1% sodium azide) was then added. After another washing step, a known amount of HRP labeled HPhA with HPhA standards or samples was added. After washing away the unbound materials, the TMB substrate was added and the color developed was reversely proportional to the amount of antigen from standards or samples. The resultant CV and RE in ELISA were within 15% at all concentrations determined for all species, so the ELISA had satisfactory accurary and precision and was an appropriate assay method to examine the pharmacokinetics of HPhA. The ELISA was suitable for measuring the compound over long periods of time and may provide an alternative technique for monitoring drug levels in clinical samples. The assay was used to determine the pharmacokinetics of HPhA in mice.The pharmacokinetic results of the HPhA after i.m. injection in rats showed that the plasma concentration-time course fitted a two-compartment open model. The pharmacokinetic results of the HPhA after i.v. injection in rats showed that the plasma concentration-time course fitted a two-compartment open model. The short half-life of theα?phase indicated that the HPhA was rapidly distributed from the central to the peripheral compartment after injection.We investigated the immunogenicity of the recombinant archaeal histone (HphA) as a non-viral gene delivery in BALB/c mice, which provided a good base for safe and effective application of HphA. And 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 cellullar immunologic response was evaluated with stimulation indexes and levels of secreted IFN-γof splenocytes. Results showed Mice immunized with HphA produced no specific antibody, and the stimulation indexes and levels of IFN-γhad no difference from the control group. The HphA had no immunogenicity in mouse,and could not produce humoral and cellular immune response.In this article, HPhA proved to enhance the in vitro and in vivo efficiency of p53 gene transfer and is a promising new strategy for p53 gene therapy. Pharmacokinetic and safety analysis of HPhA will give us information about the events occurring in the body following administration, and it is very powerful tool to make significant contribution in the rational design of HPhA mediated gene therapy. All demonstrates that HPhA may serve as a promising, wildly applicable and highly efficient tool for gene delivery and gene therapy.