| Oncolytic virus is one of the most promising therapeutic strategies for malignant tumors.Oncolytic virus can be used as a vector for gene therapy.After genetic engineering,oncolytic virus can lyse tumor cells by specific replication in tumor site,and can further destroy tumor cells by expressing therapeutic genes during replication.Oncolytic adenovirus has been widely used in clinical research due to its advantages of wide host infection,high transgenic efficiency,safety and easy production.However,oncolytic adenoviruses still have some application limitations in gene therapy:First,oncolytic viruses cannot be administered intravenously because of insufficient viral targeting,severe hepatic enrichment,and their inability to penetrate physiological barriers,thus limiting their efficacy in the treatment of gliomas and others;secondly,the virus is highly immunogenic,and it is easy to arouse anti-viral immunity against the virus in the body,but the anti-tumor immunity that can be caused is limited.In order to improve the targeting ability of oncolytic adenoviruses to achieve intravenous systemic administration,researchers have used various strategies to modify adenoviruses.Our research group also carried out a series of genetic engineering modifications against oncolytic adenovirus in the early stage and significantly improved the targeted anti-tumor effect of oncolytic adenovirus.In this thesis,we tried to further modify the oncolytic adenovirus we modified earlier by using exosomes,a biological carrier that has been a hot research topic in recent years,in order to improve its targeting and anti-tumor ability.Exosomes are extracellular vesicles that have receptor recognition capabilities similar to those of parent cells,and have attracted attention as a new type of drug carrier in recent years.In addition,exosomes are formed as part of virus information transmission during virus infection,which can load the genetic material of the virus into exosomes,and with the secretion of cells and the absorption of adjacent cells,realize the transmission of virus information and promote the virus infection.In order to improve the potential of oncolytic adenovirus targeted therapy for malignant glioma,the research group designed and constructed a capsid double modified oncolytic adenovirus(A4/K37)in the early stage.Although it can significantly improve the infection and killing activity of glioma cells,it cannot effectively break through the blood-brain barrier to achieve the purpose of two-stage brain targeting in the in situ model.Recently,some studies have reported that tumor or stem cell-derived exosomes can carry some small-molecule drugs to penetrate the blood-brain barrier,this paper designs and uses neural stem cell-derived exosomes to modify the above-mentioned genetically engineered targeted oncolytic adenoviruses,enhance the ability of oncolytic adenovirus to break through the blood-brain barrier and target glioma cells.Since the physical loading method will destroy the natural structure of exosomes,resulting in the loss of exosome content and affecting the biological function of exosomes,it is not suitable for this study,and there is almost no difference in particle size between exosomes and adenovirus,and it is not suitable for mixed incubation method.Therefore,this paper mainly attempts to isolate and obtain exosomes containing oncolytic adenovirus particles or components after virus infection,and evaluate their potential in the treatment of malignant tumors and explore related mechanisms.Firstly,we used mouse neural stem cell C17.2 as the source of exosomes,and used the process of virus infection of cells to obtain exosomes containing viral components.Conventional oncolytic adenoviruses generally have poor infectivity to neural stem cells.However,it was found by comparison that A4/K37 virus was more efficient in infecting C17.2 cells.After exosomes were collected using A4/K37 infected C17.2,density gradient centrifugation was performed and 4 density bands were collected,exosomes(Adexo)with more viral proteins and genomes were obtained and identified by screening,the results of electron microscopy,DOT BLOT and sequencing showed that Adexo had similar physical properties to exosomes(exo)secreted by C17.2 cells,including particle size,potential and Physical form,etc.,there are almost no complete virus particles inside,and it is an exosome containing components such as viral proteins and genomes.More interestingly,this exosome can infect cells like a virus,and generates progeny viruses with biological functions during the infection process to kill tumor cells,which shows that we have successfully designed and isolated an"oncolytic"exosome.After obtaining the"oncolytic"exosome,we first evaluated its targeted infection and killing ability against glioblastoma cells.The in vitro results showed that compared with A4/K37,Adexo had better infectivity and cell killing activity on human malignant glioma cells U87.In order to evaluate its in vivo effect,a mouse intracranial tumor model was established using U87 cells,and the tumor targeting ability of Adexo and its therapeutic effect on glioma were detected by intravenous administration.The results showed that compared with A4/K37,Adexo had better tumor targeting,could cross the blood-brain barrier to reach the tumor site,and also had better tumor suppressor effect,with more virus infiltration and upregulation of tumor cell apoptosis levels,Adexo extended the survival time of nude mice.Different sources of exosomes have been reported to play a role in the anti-tumor process.For example,tumor cell-derived exosomes can promote the growth and migration of tumor stem cells,while immune cells secreted exosomes can inhibit tumor growth through immune regulation.Therefore,we explored the ability of neural stem cell-derived Adexo for immune activation.Flow cytometry was used to analyze Adexo’s ability to activate macrophage line RAW 264.7 as well as primary isolated antigen-presenting cells(APCs)such as DCs and NK cells.The results showed that Adexo could effectively activate RAW 264.7,at the same time,DCs and NK also have a certain regulatory effect.Since in situ models of glioma cells are not conducive to the evaluation of immune effects,in order to evaluate the immunomodulatory function of Adexo in immunocompetent mice,we selected mouse tumor cell lines CT26 and B16 as models.Through two tumor model for short-term,compared the activation effect of Adexo and Ad(i.e.A4/K37)on immune cells in different models and with different injection methods(intratumoural and intravenous),by short-term administration of the two tumor models.The results showed that different injection methods have little effect on the types of immune cells that can be activated,and that Adexo was able to elicit more activation of intrinsic immune cells compared to Ad in both tumor models,which means that Adexo may elicit stronger anti-tumor immunity during tumor therapy.Based on the above conclusions,we hope to further evaluate the anti-tumor effect of Adexo in the immune totipotent mouse tumor model and the immunomodulatory effect in the anti-tumor process.However,since the ability of exosomes and Adexo to infect and kill two mouse tumor cells is unclear,we first evaluated the infective and killing effects of Adexo on th e two cell lines in vitro.The results showed that compared with Ad,Adexo showed better infectivity in B16 cell line,and there was no significant difference in infectivity between the two in CT26 cell line.At the same time,there was no significant difference in killing effect between Ad and Adexo against the two cell lines.Finally,we chose to further evaluate the antitumor effect of Adexo on the B16 model,and analyze its immunoregulatory mechanism on the tumor microenvironment.The results showed that Adexo has better antitumor effect and immune activation than Ad,and could increase the number of Dendritic Cells(DCs),activated CD4~+T cells and CD8~+T cells,while significantly reducing the number of intratumoral M2 TAMs.More importantly,Adexo significantly activates B cells,stimulates the occurrence of systemic immune responses,and produces tumor-specific antibodies,improving the effect of anti-tumor immunotherapy.Through RNA sequencing of Adexo and exo,we have preliminarily explored the possible molecular mechanism of B-cell activation by Adexo and hypothesized that Adexo may promote B-cell activation and anti-tumour effects through RNA.In summary,this paper obteined a neural stem cell exosome Adexo with"oncolytic"function,and draws the following conclusions by analyzing its specific properties and functions in vitro and in vivo:Adexo not only has the physical and chemical properties of exosomes derived from neural stem cells,which can penetrate the blood-brain barrier to reach the tumor site after intravenous injection,but also has a certain immune activation effect,which can activate the generation of anti-tumor immunity.it also has a"oncolytic"function,which can infect like a virus,And can generate complete,biologically active progeny viruses.In the anti-tumor process of Adexo,it first reaches the tumor site through the unique properties of exosomes and activates the innate and adaptive immune responses,and then infects tumor cells to carry out adenovirus packaging and replication,and at the same time plays the role of virus oncolysis and gene therapy effect,thus exerting a better anti-tumor effect.Through the research of this paper,the anti-tumor immune regulation function of neural stem cell-derived exosomes is further understood,and it also provides a new idea for the targeted modification of oncolytic adenovirus. |
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