| Glioblastoma multiforme(GBM)is the most common brain t?mor,and the average survival of patients is less than 15 months.Because of the brain location and the complex biological characteristics,GBM is difficult to cure using conventional cancer therapies(surgery,chemotherapy and radiothe).In recent years,oncolytic virotherapy,as a new therapeutic strategy for malignant t?mors,has been widely concerned.According to statistics,there are at least 15 clinical studies using oncolytic virus for the treatment of glioblastoma.The types of oncolytic viruses used in these clinical studies include adenovirus,herpes simplex virus,reovirus and poliovirus,among which adenovirus is the most widely used virus.Currently,oncolytic adenoviruses are developed based on adenovirus serotype 5(Ad5),also known as conditionally replicative Ad5(CRAd5).Ad5 infects cells depend on its fiber,which can bind to the coxsackie and adenovirus receptor(CAR)on the cell surface to anchor the virus on the cell.However,CAR is usually lowly expressed in glioma cells,and the expression of CAR in normal tissue cells is generally higher than that in glioma cells.This may lead to poor targeting infectivity of oncolytic virus to glioma cells,which severely limits the application of oncolytic virus in glioblastoma therapy.In addition,due to the existence of blood-brain barrier and the limitation of virus infection mechanism,oncolytic viruses,involving oncolytic adenoviruses used in clinical studies,are generally intrat?morally administrated to treat glioblastoma.In this case,virus cannot spread to infect glioma cells far away the injection sites.Therefore,Oncolytic viruses cannot efficiently inhibit the t?mor progression for glioblastoma therapy by intrat?moral injection,as glioblastoma is prone to occur in multisites,aggressive and easy to recur and metastasize.Based on these,the aim of this study is developing adenovirus vectors,which can efficiently infect glioma cells and be administrated via systemic administration,through modifying the adenovirus capsids.To date,dozens of serotypes of h?man adenoviruses have been found,belonging to seven subgroups.The infection mechanism of adenovirus infection is different in different subgroups,for example,Ad5 from subgroup C depends on CAR to infect cells,while Ad37 from subgroup D infects cells through sialic acid(SA).Sialic acid is an important molecule in the process of neurodevelopment,which is expressed in large quantities in the brain.And it has been reported that the abundance of sialic acid is related to the malignancy of t?mors.Therefore,we hypothesize that if the knob region of CRAd5 is replaced by knob(Ad37-knob,K37)of Ad37,the fiber chimeric oncolytic adenovirus may alter the infective nature of oncolytic adenovirus and enable it to infect cells through sialic acid as a receptor.the chimeric virus will have the potential to infect and dissolve more efficiently.In addition,this strategy is expected to enhance the targeting of oncolytic virus towards blood-brain barrier and glioma.The first part of this paper focuses on this hypothesis.After the construction of virus skeleton plasmid,virus packaging,screening and purification,we successfully obtained the Ad37-knob exchanged oncolytic adenovirus(CRAd5/K37).The in vivo and in vitro results showed that Ad37-knob replacement strategy could change tropism of oncolytic adenovirus,and proved that this strategy was able to significantly enhance the ability of oncolytic virus to infect glioma cells(the increase more than ten times).Then,we evaluated the brain-blood-barrier(BBB)ability of CRAd5/K37.The results showed that CRAd5/K37 could cross the in-vitro BBB model more efficiently than CRAd5 without modifications of the capsid,while CRAd5/K37 did not acc?mulate in the brain of mice bearing intracranial t?mors and normal Balb/c mice,when administrated via intravenous injection.There are two possible reasons for this phenomenon:(1)The expression of sialic acid in mouse-derived cells is significantly lower than that in the corresponding h?man cells,which may lead to the inconsistency between the conclusion of in vitro and in vivo experiments.Mouse model may not be an appropriate animal model for evaluating tissue distribution of Ad37-knob replacement adenovirus.To further clarify whether Ad37-knob replacement strategy can effectively enhance the transversing ability of oncolytic adenovirus,and analyses in other advanced animal models(e.g.monkey)are therefore needed to better evaluate this potential.(2)A single knob replacement strategy may not be enough to significantly enhance the brain targeting ability of oncolytic virus,and it may be possible to further achieve brain targeting of oncolytic virus through additional modifications.In the second part of this paper,we made efforts to insert targeting peptides into the knob region of CRAd5/K37,in order to further enhance the dual-targeting of oncolytic adenovirus towards BBB and glioma cells.Five brain targeting peptides were selected,which have been successfully applied to mediate non-viral vectors or other viral vectors targeted to brain.Referring to the targeting-peptide insertion sites on Ad5-knob,10 modified forms of Ad37-knob(K37-peptide)with targeting-peptide insertions in HI loop or C-terminal were designed.However,it was found that the expression efficiency of all K37-peptide proteins was markedly lower than that of K37 protein without targeting peptide modification in Escherichia coli.Four pAd5/K37-peptide adenovirus skeleton plasmids were successfully constructed,but no virus could be generated when using them and the shuttle plasmid for virus packaging in HEK 293 cells.Finally,chimeric fibers(F5/K37 and F5/K37-peptide)expression plasmids were constructed.After transfection into eukaryotic cells,the expression of all F5/K37-peptide was far lower than that of F5/K37.So far,we deduced that the HIloop and C-terminal of Ad37-knob may do not allow the insertion of targeting peptides,and the detailed reasons and mechanisms need to be further studied in the future.In addition to fiber,adenovirus capsid protein ?(p?)is also an ideal site for adenovirus capsid modification.In our previous study,we constructed a t?mor necrosis factor-related apoptosis-inducing ligand(TRAIL)-modified oncolytic adenovirus vector,termed A4.In A4,TRAIL was linked on p? via the dimerization of leucine zipper.A4 exhibited good t?mor targeting and anti-t?mor effect in animal models of breast cancer.The third part of this paper is to construct a novel double-modified oncolytic adenovirus(A4/K37)by combining TRAIL surface modification and Ad37-knob replacement strategies,in order to make the oncolytic adenovirus have better infection ability and selectivity to glioblastoma.A4/K37 virus was successfully obtained through virus packaging,screening and purification.Then,the genome and structural properties of A4/K37 were identified,and its infection properties in vitro were analyzed.Finally,the anti-t?mor effect of A4/K37 virus was evaluated in t?mor-bearing mice.From analyzing all these results,it is concluded that the dual-modification strategy can optimize the infection and selectivity of the virus to glioma cells,and A4/K37 has better antit?mor effect than A4 virus.Double modification strategy can achieve more efficient replication oncolysis and more effective TRAIL gene therapy,compared with the single modification strategy.In s?mmary,this study totally evaluated three capsid modification strategies involving "Ad37-knob replacement","Targeted peptide modification based on Ad37-knob exchagement" and "dual-modification strategy of Ad37-knob replacement combined with TRAIL modification on p?",in order to enhance the potential of applying oncolytic adenovirus in glioblastoma therapies.From the results,It is concluded that "Ad37-knob replacement strategy" could modify the tropism of oncolytic adenovirus and make it more suitable for the treatment of glioblastoma;the strategy of "targeting-peptide modification on Ad37-knob exchanged oncolytic adenovirus" was not feasible,as the expression of Ad37-knob protein was seriously affected after modification,resulting in difficulties of virus packing;The oncolytic adenovirus vector constructed by "dual-modification strategy" has more efficient ability to replicate in glioma cells and is able to mediate better therapeutic effect of TRAIL gene,showing potential in future application for glioblastoma therapy. |
PDF Full Text Request