| Cancer,the second leading cause of death,is the most serious public health problem facing mankind.The main treatments for cancer currently include traditional treatments based on radiotherapy and chemotherapy,and nascent tumor treatments based on immunotherapy.The American Association for Cancer Research identified oncolytic viral therapy as the new immunotherapy approach after radiation,chemotherapy,surgical resection and targeted therapy in its 2019 annual anti-tumor report.Oncolytic viral therapy has become an effective means of cancer clinical treatment with great potential,which abandons the risk of large wound and easy infection caused by traditional surgical treatment,drug resistance and obvious side effects in the course of chemotherapy,and extreme damage to normal cells in the course of radiotherapy.However,the number of oncolytic viruses currently used in clinical is very small and is dominated by DNA viruses.The discovery of new and excellent oncolytic viruses is of great significance to both the oncolytic virus system and cancer therapy.In this study we identified two highly promising RNA oncolytic viruses,the live attenuated virus WNV-poly(A)and the propagative virus-like vesicles VEEV-delC.About 60 years ago,scientists discovered the oncolytic potential of WNV to inhibit the growth of several types of tumors,but subsequent research was terminated by the apparent toxicity after viral infection.In order to improve the biosafety of WNV oncolytic therapy,the development and application of live attenuated virus as an oncolytic virus has great clinical potential.In our previous work,we found that WNV-poly(A)viruses obtained by replacing the VR and DB structural domains in the 3’UTR of the WNV wild type virus genome with poly(A)sequences were highly attenuated.In this study,we found that WNV live attenuated virus WNV-poly(A)had significant and broad-spectrum oncolytic effect in a series of mouse xenograft tumor models and human-derived tumor models(HDST,PDX).Further studies on its oncolytic mechanism revealed that WNV-poly(A)selectively infects and kills tumor cells in a IFN-(40)-dependent manner without harming normal cells on the one hand.On the other hand,WNV-poly(A)can activate DCs to present tumor antigens and trigger CD8+T cell-mediated tumor antigen-specific responses,thus inhibiting the proliferation of primary and distant tumor cells.In addition,intravenous injection of WNV-poly(A)also significantly delayed the progression of hepatocellular carcinoma xenografts as well as the therapeutic effect of intratumoral injection.Due to the feature of self-replication and natural immunostimulatory,alphaviruses RNA are widely used in cancer therapy research based on the delivery of recombinant particles,naked or nanoparticle-encapsulated RNA or plasmid-based DNA replicons.However,these forms of delivery generally difficult to meet the requirements of actual production.In this study,we constructed an alphavirus propagative virus-like vesicles(VLVs)platform for tumor therapy.Alphavirus VLVs are capsid-free particles formed by transfection of alphavirus replicons-envelope proteins RNA.By passaging in BHK-21 or VERO cells,high titers of alphavirus VLVs can be obtained.Through this platform,the VEEV-delC replicon plasmid expressing the envelope proteins of VEEV was constructed and the self-amplified VEEV-delC VLVs particles were rescued successfully.Virological analysis showed that VEEV-delC VLVs are similar to cell microvesicles in morphology and size,and can self-amplify with titres up to 1?10~7PFU/m L after passaging,which has the potential for rapid amplification and large-scale production.In addition,VEEV-delC VLVs could infect tumor cells to release a large amount of encapsulated genomic RNA,which effectively activates the inflammatory response within tumor tissue and ultimately exerts a broad-spectrum anti-tumor effect.VEEV-delC was also found to be an expression vector for the expression of exogenous genes.Using this strategy,we constructed VEEV-delC-GMCSF VLVs with more significant oncolytic effect.This study shows that both the live attenuated virus WNV-poly(A)and the propagative microvesicles VEEV-delC are excellent and broad-spectrum oncolytic viruses,which could effectively reverse the immunosuppressed tumor microenvironment.These characteristics give them great potential for clinical development and promise to be clinical anti-tumor treatment strategies and ultimately against metastatic and recurrent tumors. |
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