| Cancer is one of the major diseases which seriously threat human life and health,Safe and effective treatment for cancer is currently the world’s health problems and social problems to be solved.With the deep development of life science and medical research,biological treatment has become the fourth largest category for cancer treatment behind the surgery,radiotherapy and chemotherapy.Gene therapy based on adenovirus vectors is an important direction of the current biological therapy for malignant tumors.In addition,oncolytic adenovirus,due to a wide infection host,safe and easy production,larger exogenous genes can be inserted into,etc.have also been widely studied and applied.Combining the dual advantages of gene therapy and oncolytic virotherapy,the oncolytic adenoviral gene therapy is a very promising strategy for cancer treatment.However,with clinical studies about adenoviral vectors deepening,people found that there are still many limitations to the further use of adenoviral vector in the human body,such as the pre-existing anti-vector immunity,in vivo liver enrichment by intravenous administration,and inadequate targeting and anti-tumor activity,etc and need to be further improved.In this thesis,by selecting the appropriate specific tumor therapeutic gene,and using molecular biology techniques to modify oncolytic adenovirus capsid protein to regulate their biological properties and tumor suppressor activity,we hope to obtain the new oncolytic adenoviral gene therapy vectors with targeting,security and stronger anti-tumor activity,provide the basis for human clinical applications with adenoviral vectors and cancer gene therapy researchCurrently targets for adenovirus gene that can be modified focused on three kinds of major capsid protein:protein fiber(fiber),penton base(penton),hexon(hexon),as well as the minor capsid protein pⅨ.Due to the important effect of the virus structural protein for stability and assembly of viral capsids,also because genetically modified capsid protein,such as fiber protein,often affect the packaging and infection efficiency of virus,therefore the goal of the transformation of the paper is mainly pⅨ Structural analysis showed that,pⅨ on each side of adenovirus capsid connect and form the four-trimer network.In this model,the three C-terminal of the four-dimerization exposed,and the fourth anti-parallel Tumor necrosis factor-related apoptosis-inducing ligand,TRAIL through death receptor pathway can specifically induce a variety of tumor cell apoptosis and non-toxic to normal cells.Not only recombinant TRAIL protein has a strong anti-tumor activity,TRAIL gene transduced into tumor cells for gene therapy can also be effective in inducing tumor cell apoptosis through the using of viral vectors and non-viral vectors.In addition,the recombinant form of TRAIL by intravenous or intraperitoneal administration also showed its potential with certain targeted tumor.Therefore,in this essay,we utilize a leucine zipper heterodimers system,fuse one of the single zipper chain to the C-terminus pⅨ,and also fuse the paired zipper to the N-terminal of an oncolytic adenovirus expressed TRAIL,in this way trimeric TRAIL produced by vectors will be modified to the surface of capsids that completely assembly by Zipper pairing.Features of the carrier is that modify pⅨ with only a short peptide,so it will not affect the virus packaging;TRAIL directly expressed by vector can induce tumor apoptosis and will also be connected to the capsid protein of the stable progeny virus,which influence polyomavirus biological properties and increase their stability so as to obtain a stronger tumor suppressor activity.In this paper,after the genetic modification of backbone plasmid and the construction of the relevant shuttle vector,we co-transfect both plasmids into 293 cells and then successfully packaged a pⅨ modified adenoviral.Followed by CsCl density gradient centrifugation and immune electron microscopy,we confirm that the surface of the purified adenovirus indeed exist TRAIL protein.To further analyze the biological activity of TRAIL on the viral surface,we detect the inhibitory activity of viral vectors against tumor cells at different stages of adenovirus infection.Comparison of the unmodified vector,we found pⅨ modified vectors in the early viral infection(within 6 hours)have better apoptotic activity for tumor cells,and the performance of magnificant a more significant anti-tumor activity after the infection continued until 72 hours,which suggest that TRAIL protein connected to the adenovirus surface not only maintain its biological activity,but also significantly enhances the oncolytic adenovirus vector antitumor activity.Conventionally the route of administration of adenoviral vector is intratumoral injection,although have a high local drug concentration,but the diffusion is limited,and for the larger tumor it needs more injection sites,which,increase the number of operations and the distribution is uneven.And most malignant surgery or recurrence after radiotherapy is multi-center,so the direct intratumoral injection of the virus is not appropriate.Systemic administration of adenovirus vectors have not yet been widely accepted,primarily because the virus using such administration tends enrich in liver,and rarely can reach the primary site and the metastases.In this paper,we utilize red fluorescent protein-RFP and small animal in vivo imaging system,which show that after intravenously injected pⅨ modified oncolytic adenovirus for 4 days,the expression of RFP in tumor-bearing mice can be detected in the tumor tissue indicating that the TRAIL-carrying oncolytic adenovirus with modified pⅨ gain tumor targeting property.Subsequently,we carry on deep analysis for anti-tumor activity of pⅨ modified oncolytic adenovirus.In vitro,pⅨ-modified oncolytic adenovirus have good anti-tumor effect for a variety of tumor cells(ZR-75-30,MCF-7,Hela,MGC80-3,APNC-1,HepG-2,etc.),but no toxic side effects in normal cells(MCF-10A,CCD-11Lu,QSGC-7701,etc.).In vivo we firstly established mouse model of breast tumor cells ZR-75-30,and carry on intratumoral injection and intravenous injection for tumor-bearing mice with recombinant adenovirus to evaluate the effect of cancer treatment.Compared to conventional oncolytic adenovirus,modified rAd5zp-zTRAIL-RFP-SΔ24E1a virus exhibited more significant antitumor effect with either intravenous or intratumoral admininstration and can be able to detect significant caspase-3 activity in tumor tissue after two weeks’ treatment.Meanwhile,HE staining experiments showed pⅨ modified oncolytic adenovirus vectors will not cause damage or toxicity in mice’ s liver and kidney tissue regardless of the mode of administration,which showed a good safety.In summary,we successfully obtained a novel targeted oncolytic adenoviral gene-therapy vector that can be administered intravenously,and under TRAIL’s selective guide blood circulation will transport oncolytic adenovirus to the tumor tissue,play gene therapy and viral oncolysis.The studies of this dissertation provide a good foudnation for further study and applications of oncolytic adenoviral gene-therapy vectors. |
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