Construction Of Oncolytic Adenovirus Targeting Tumor Vasculature And Analysis Of Its Infection

Oncolytic therapy is a new treatment in tumor biotherapy applications,which uses the virus’ s replication ability to selectively infect and lyse tumor cells while retaining normal cells and tissues.Progeny viruses and tumor-associated antigens can be released by Lysed tumor cells,which stimulate the body to produce an immune response to further kill tumor cells.In addition,studies have shown that some OVs(such as oncolytic pox virus)can specifically infect and destroy tumor blood vessels,indirectly causing apoptosis or necrosis of uninfected tumor cells.Tumor neo-vasculature provides oxygen and nutrients for tumor cell proliferation and metastasis.OVs will further enhance its tumor-killing ability and inhibit tumor invasion and metastasis through targeted infection and destruction of tumor blood vessels.Oncolytic adenovirus,also known as conditionally replicating adenovirus(CR Ad),is one of the most widely used oncolytic viruses in clinical research.The oncolytic adenovirus currently under development is mainly based on the modification of human adenovirus serotype 5(Ad5).Coxsackie-Adenovirus Receptor(CAR)is main cellular receptor of Ad5.Adenovirus binds to the receptor through its fiber,which attaches to the cell surface and internalizes using the integrin receptor.The low or no expression of CAR on the surface of tumor vascular endothelial cells makes CRAd5 lack the ability to infect tumor vascular endothelial cells,thus limiting the potential of oncolytic adenovirus to enhance its antitumor effect by destroying tumor blood vessels.Currently,more than 60 serotypes of human adenovirus have been found and divided into seven subgroups of A-G.Due to different recognition receptors,different serotypes of adenoviruses infect different cell types.Such as,the main serotype cell receptor of Ad5,which from C subgroup,is CAR,while Ad37 of D subgroup uses sialic acid(SA)as the receptor.Sialic acid is an acetylated derivative of neuraminic acid.The content of sialic acid is closely related to the increase,metastasis,and infiltration of tumors.And different forms of sialic acid are related to the adhesion of vascular endothelial cells.Therefore,we propose the following hypotheses: 1.The sialic acid receptor on the surface of tumor vascular endothelial cells will exhibit high expression.2.Replace the knob region of the Ad5 with the Ad37-knob to construct a fiber chimeric virus,which may enhance the ability of CRAd5 to infect tumor vascular endothelial cells.In addition,studies have reported that RGD-modified oncolytic viruses can enhance targeting and antitumor activity in tumor models with high integrin expression.At the same time,RGD peptides compete for extracellular matrix integrin receptors,inhibit tumor angiogenesis,and reduce the ability of tumor cells to infiltrate and adhere to normal tissues.Therefore,modifying the fiber chimeric virus with RGD will further enhance the ability of CRAd5 to target tumor blood vessels.Based on the above background,this paper designs and constructs a new type of fiber chimeric oncolytic adenovirus to enhance the ability of oncolytic adenovirus to target tumor vascular endothelial cells.First,replace the knob of CRAd5 with Ad37-knob to construct CRAd5/K37;then,insert or mutate the RGD peptide in the knob or other fiber region of CRAd5/K37 to further construct the RGD-modified chimeric oncolytic adenovirus CRAd5/K37-RGD.After obtaining the above chimeric virus,its ability and properties to infect tumor cells and tumor vascular endothelial cells were analyzed in depth.This paper first analyzes the feasibility of oncolytic adenovirus modification strategies.Flow cytometry revealed that sialic acid receptors on the surface of human umbilical vein endothelial cells(HUVEC)showed high expression.Before constructing the viral vector,two chimeric fiber expression vectors with two modification strategies were first constructed,and the expression of the chimeric fiber and the trimeric form were analyzed by Western Blot.The results showed that the chimeric fiber(F5/K37)was able to express normally and form trimers;the insertion of the RGD peptide in the HI-loop loop of the Ad37 knob region destroyed the expression of the chimeric fiber and the formation of trimers,and thus affected the virus packaging;Mutation of the RGD peptide in the fiber’s shaft region does not affect the expression of the chimeric fiber and the formation of trimers.Subsequently,based on the above preliminary verification,chimeric oncolytic adenoviruses CRAd5/K37 and CRAd5/K37-S-RGD were constructed with tumorspecific promoter Survivin to mediate E1 gene expression.The above two new oncolytic adenoviruses were successfully obtained through the construction of adenovirus backbone plasmid and the steps of virus packaging,screening,purification and titer determination.Then,the type change of the chimeric viral fiber was verified by experiments.Next,we focused on the infectious properties of chimeric oncolytic adenovirus on human vascular endothelial cells and tumor cells.HUVEC,human microcerebra vascular endothelial cells(hCMEC/D3),normal hepatocytes,and various types Tumor cell lines were analyzed by flow cytometry.The results showed that CRAd5/K37 and CRAd5/K37-S-RGD had significantly improved infection capabilities in HUVEC and various tumor cell lines compared to the unmodified virus CRAd5.Comparing the two chimeric viruses,although CRAd5/K37-S-RGD failed to improve infection capabilities in HUVEC and various tumor cell lines compared to CRAd5/K37,it is more selective for tumor cells and tumor vascular endothelial cells,indicating that CRAd5/K37-S-RGD may have better security.Then,in SKOV3 model,blockade by the knob protein further clarified the main receptors dependent on CRAd5/K37-S-RGD,incuding CAR,SA,integrin and so on.We further analyzed the inhibitory ability of oncolytic adenovirus to tumor vascular endothelial cells by flow cytometry,RT-PCR,MTT and other methods.The chimeric viruses CRAd5/K37 and CRAd5/K37-S-RGD can significantly inhibit the cell viability of HUVEC.At the same time,due to the improvement of the ability to infect tumor cells,both chimeric viruses can significantly inhibit VEGF,which secreted tumor cells to enchance tumor vascular abnormalities.Finally,by simulating the environment of chimeric oncolytic adenovirus infection in vitro,hVEGF165 can significantly enhance the ability of CRAd5/K37-S-RGD to target tumor endothelial cells.Finally,we established a mouse tumor-bearing model with the human ovarian cancer cell line SKOV3,and confirmed by immunofluorescence experiments that the chimeric viruses CRAd5/K37 and CRAd5/K37-S-RGD can efficiently infect mouse tumor vascular endothelial cells.Subsequently,we established a mouse tumor-bearing model with the mouse ovarian cancer cell line OVHM,and analyzed the ability of chimeric oncolytic adenovirus to inhibit tumor growth by destroying tumor blood vessels.The two chimeric viruses did not significantly improve the tumor growth inhibitory ability of CRAd5.Analyzing the reasons,we believe that because human adenovirus cannot effectively replicate in mouse-derived cells,the simple increase in infectivity failed to cause sufficient damage to tumor blood vessels and significantly inhibit the growth of tumor cells.In summary,this paper successfully constructed a new oncolytic adenovirus with the ability to target tumor blood vessels through fibrin transformation,providing a good basis for the research and application of oncolytic adenovirus vectors in anti-tumor angiogenesis gene therapy.