TRAIL Modified Oncolytic Adenovirus Vector In Acute Myeloid Leukemia Treatment

Acute myeloid leukemia(AML)is a heterogeneous malignant tumor of the blood system,which poses a serious threat to human health.At present,the clinical treatment of AML is still based on radiotherapy and chemotherapy,bone marrow transplantation.Despite the progress of treatment,the overall survival rate of AML patients is still very low.Drug resistance to first-line chemotherapeutic drugs is the main cause of treatment failure,so it is the primary task of AML treatment to continue to seek new treatment methods.With the further development of life science and medical research,Oncolytic virus has become an important development direction in the treatment of malignant tumors.However,due to the lack of targeting and infection efficiency,the use of oncolytic virus in the treatment of hematological malignancies such as leukemia is still limited.Oncolytic adenovirus is one of the most widely used viral types in the treatment of malignant tumors.However,limited by the way of administration(currently mainly through intratumoral administration),there are few studies on oncolytic adenovirus in the treatment of hematological malignancies.In our previous research,we successfully constructed a targeted adenovirus vector(rAd5pz-zTRAIL-RFP-S24E1 a,A4)by structural modification of adenovirus using leucine zipper isodimer system.The vector can be used intravenously to treat breast cancer in mice.Therefore,this paper hopes to optimize the vector and further investigate its potential in AML treatment.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)can be expressed by oncolytic adenovirus A4,and it can be linked to the virus surface by leucine zipper,thus targeting cancer cells and enhancing tumor killing ability.Therefore,the expression levels of adenovirus receptors and TRAIL receptors on AML cell lines and primary cells were detected.The results showed that high levels of death receptors DR4 and DR5 were expressed in immortal AML cell lines THP-1 and MV4-11,but low levels of deception receptors(DcR1 and DcR2)were observed.Adenovirus receptors(CAR,integrin alpha v beta 3 and integrin alpha v beta 5)were expressed in both cells.In 19 AML patients tested,more than 50% of the samples expressed moderate levels of DR4 and DR5.This result lays a foundation for the application of recombinant oncolytic adenovirus vector.This suggests that A4 may be able to achieve targeted therapy for AML.Previous studies have found that although the capsid surface of A4 virus is modified with TRAIL by zipper,the amount of TRAIL modified by the capsid of A4 virus is less than the theoretical value.Therefore,in order to further enhance the tumor targeting ability of A4,we first constructed the prokaryotic expression plasmid pET-28a-z-sTRAIL,a truncated TRAIL fusion protein with C-terminal leucine zipper E.E34 single chain,and successfully expressed and purified the z-sTRAIL protein in E.coli.After verifying the basic biological activity of z-sTRAIL,z-sTRAIL protein was co-incubated with adenovirus capsid pIX modified with zipper R.R34 single-stranded virus in vitro and purified by CsCl density gradient centrifugation.The dot blot and ELISA results showed that the recombinant oncolytic adenovirus vector zA4 with twice the TRAIL modification of A4 capsid could be obtained by our optimized method in vitro.Before analyzing the infection and replication ability of recombinant oncolytic adenovirus vectors A3,A4 and zA4 in AML cells,we first examined the integration of TRAIL receptors(death receptors,DR4 and DR5;bait receptors,DcR1 and DcR2)and adenovirus receptors(Coxsackie adenovirus receptors,CAR)in AML cell lines THP-1,MV4-11 and primary AML cells.The expression of integrin alpha v beta 3 and integrin alpha v beta 5.Flow cytometry showed that both immortal AML cell lines THP-1 and MV4-11 expressed higher levels of death receptors DR4 and DR5,but lower levels of deception receptors(DcR1 and DcR2)and lower levels of adenoviral receptors(CAR,integrin alpha v beta 3 and integrin alpha v beta 5).In 19 AML patients tested,more than 50% of the samples expressed moderate levels of DR4 and DR5.This result lays a foundation for the application of recombinant oncolytic adenovirus vector.Through the infection analysis of THP-1 and MV4-11 cells,we found that under the same conditions,with the increase of the amount of TRAIL protein modified by adenovirus vector capsid,the infection ability of the virus vector also showed an increasing trend.Furthermore,the binding and internalization experiments of THP-1 with virus further proved that the more TRAIL proteins modified on the surface of virus vector,the better the binding ability of virus vector to THP-1 cells.By evaluating the anti-tumor effect in vitro,we can find that different MOI viruses infect THP-1 and MV4-11 cells respectively,and all three oncolytic adenoviruses can induce dose-dependent AML cytotoxicity.It has no obvious killing effect on human normal lymphocyte T cells.The same cytotoxicity analysis was carried out on primary AML cells from 19 AML patients with the same method.The results showed that all three oncolytic adenoviruses could kill primary AML cells significantly,and the killing ability increased with the increase of TRAIL protein modification in the viral capsid.Balb/c nude mice were used to evaluate the anti-tumor effect in vivo.We successfully established two kinds of AML models: subcutaneous and venous tumor models.In both models,the three viruses have obvious tumor targeting effect and tumor inhibition effect,and the effect of zA4 is the most obvious.In order to further enhance the killing ability of recombinant oncolytic adenovirus zA4 in tumor cell lines expressing relative death receptor of TRAIL,ginsenoside Rh2 was used to enhance the expression of death receptor of tumor cells,so as to enhance the apoptotic activity induced by TRAIL.It was found that low dose of Rh2 and low dose of Rh2 were used simultaneously.When combined with sTRAIL,IC50 of sTRAIL could be reduced,and a strong synergistic effect was found between them.Then we used immortalized AML cell line THP-1 and primary AML cells to evaluate the anti-tumor effect of Rh2 combined with zA4 in vitro.It was found that Rh2 could significantly enhance the anti-tumor ability of zA4 in both AML cells.At the end of this study,we evaluated the synergistic effect in vivo using the intravenous tumor-bearing model.The experimental results show that the combined therapy can effectively inhibit the proliferation of tumor cells and liver infiltration,and effectively prolong the survival period of mice.In conclusion,in this study,we have obtained more TRAIL protein modified oncolytic adenovirus vector zA4,which has been proved to have good anti-tumor activity at the cell level in vitro.In addition,intravenous injection of zA4 in nude mice tumor-bearing model shows better tumor targeting and killing.Injury ability.In addition,the combination of Rh2 and zA4 enhances the targeted therapeutic effect of zA4 on primary AML cells and transplanted AML nude mice models.This study provides the feasibility of oncolytic adenovirus vector in the treatment of hematological malignancies including AML.