Preparation Of Lipid-based MRNA Nanovaccine And Its Application In Colon Cancer Immunotherapy

Background and objective:Colorectal cancer(CRC)is one of the most common cancer types in the world,also the third leading cause of cancer death.The research of treatment for colorectal cancer has already become one of the most crucial health projects worldwide and the need for an efficient and novel therapeutic strategy is urgent as well.Nowadays,apart from traditional treatment:surgery,chemotherapy,and radiotherapy,cancer immunotherapy has already become an essential treatment method and research field for CRC.Cancer vaccine,as a therapeutical biological administration for cancers,has become an essential part and hotspot because of the ability to generate specific antitumor immune responses by harnessing and adjusting the immune system,thus inhibiting tumor progression and metastasis successfully.In the meanwhile,cancer vaccines are proficient,reliable safe,and easy to modify.mRNA vaccine has bloomed to manifest promising effects in multiple types of diseases including cancer.However,the therapeutic mRNA vaccine for CRC still needs further investigation.Besides,the function of the mRNA vaccine is influenced by many factors,and multiple inflammatory pathways,such as STING,TLR,RIG-I pathways are confirmed to be influential.Nevertheless,the mechanism and pathways involved in the process of vaccination are remained to be uncovered.In this study,we systematically design and screen an mRNA nanovaccine based on the cationic lipid EDOPC.The mRNA nanovaccine exploits the specific antitumor therapeutic effect for colon cancer by inducing efficient innate and adaptive immune responses.In addition,we are going to uncover the inflammatory pathways and regulatory factors in the immunization course of mRNA nanovaccine.We wish this study can provide new ideas and insights for the future design of mRNA cancer vaccines and immunotherapy for cancers.Methods:1.The design and screen for mRNA nanovaccine:(1)In order to systemically design and screen the mRNA nanovaccine,we tested the ratio and dosage of each component in our formulation in our study.In this way,the mRNA nanovaccine is able to protect the mRNA molecules and shows great stability.In our study,nucleotide gel-retardation was performed to find the proper ratio for mRNA to protamine and RNA quantification assay was used to test the encapsulation efficiency for the mRNA-protamine core of the vaccine particle.The zeta-size instrument was employed to measure the size and superficial charge of the vaccine particle.Transmission electron microscope was applied to visualize the morphology of the mRNA nanovaccine.(2)To enable the cationic liposomal mRNA nanovaccine to have satisfying and high transfection and expression efficiency in DC.In order to measure the transfection efficiency of mRNA nanovaccine candidates,reporter mRNA that express GFP or luciferase were loaded and delivered into murine-derived DC and assessed by flowcytometry or spectrophotometry.The cytotoxicity of the mRNA nanovaccine was tested in BMDC by MTS assay.In addition,the encapsulation and expression of lyophilized mRNA nanovaccine were also evaluated.2.Antigen presentation and T cells activation of mRNA nanovaccine:(1)To assess the ability to present the target antigen and induce the immune response in vitro situation.BMDC were harvested and treated with OVA mRNA nanovaccine.The surface marker of maturation and OVA-MHC I complex level were evaluated by flowcytometry.(2)By co-culturing the activated BMDC with T cells,the T cell proliferation was detected by flowcytometry.IL-2 secretion of activated T cells was also measured by ELISA after 24 hours of stimulation.3.The biosafety and expression in vivo of mRNA nanovaccine:The BALB/c mice were immunized with Luciferase mRNA nanovaccine in the footpad.The biosafety was tested by monitoring the body weight change 5 days after the injection.The fluorescence signal of luciferase in vivo was detected by the IVIS system.4.T cells activation in vivo and tumor inhibition effect of mRNA nanovaccine:(1)In order to study the biological activity and tumor inhibition ability of mRNA vaccine in vivo situation.The single cell suspension was prepared by extracting the p LN after footpad injection in C57BL/6 mice.The maturation level of DC subunits and the proportion of T cells were analyzed by flowcytometry.The frequency of IFN-γ~+cells was measured by ELISPOT assay.(2)To evaluate the capability of mRNA nanovaccine to inhibit murine colon cancer growth.We subcutaneously injected the MC38-OVA cells in the left flank of the mice abdomen and immunized the mice with OVA mRNA nanovaccine 3 and 7 days after the inoculation of tumor cells.Tumor growth was measured every 2 days.SPL,draining p LN,and tumor were harvested on day 15 and prepared for the single cell suspension.IFN-γ~+CD8~+T cells and OVA-MHC I antigen-specific CD8~+T cells by dextramer were tested by flowcytometry.In addition,the frequency of IFN-γ~+cells from SPL and p LN were visualized by ELISPOT assay.5.Immune responses and mechanism of mRNA nanovaccine:(1)BMDC were treated with mRNA nanovaccine for 24 hours and the cytokines,including IFN-β,TNF-α,IL-1β,CCL5 in the cell culture medium were measured by ELISA assay.(2)To uncover the potential mechanisms involved in mRNA nanovaccine,we treated BMDC from genetically engineered mice which were STING,TRIF,TLR7,and MAVS-KO with mRNA nanovaccine for24 hours and compared the secretion of IFN-βand TNF-αwith WT mice.To elucidate the role of each component in the formulation,EDOPC was replaced with cationic lipid DOTAP or neutral parental lipid DOPC,and the IFN-βand TNF-αlevels were tested in WT,STING-KO,MAVS-KO BMDC by ELISA assay.The knockout effect of STING and MAVS was detected by western blot.The phosphorylation of TBK1,IRF3,IRF7,P65the downstream essential marker for STING,TLR7 and MAVS activation was detected by the western blot.6.The impact of STING and MAVS pathways in mRNA nanovaccine:(1)To compare the impact of the STING pathway of mRNA nanovaccine with WT mice,p LN of WT and STING-KO mice were harvested after vaccination with our vaccine,and the maturation of DC and activation of T cells were measured by flowcytometry.To compare the impact of the MAVS pathway of mRNA nanovaccine with WT mice,p LN were collected after vaccination.Frequency of CD44~+CD8~+memory T cells and antigen-specific OVA-MHC I CD8~+T cells were tested.In addition,the frequency of IFN-γ~+cells from SPL and p LN were tested by ELISPOT assay.(2)Based on the findings above,the therapeutic effect was examined by MC38-OVA tumor model in WT and STING-KO mice.IFN-γ~+CD8~+T cell frequency was measured in p LN and tumor tissues.Results:1.In our study,after the systematic design and screening,the formulation of the mRNA nanovaccine was selected.In accordance with our results,our mRNA nanovaccine possessed good characteristics of mRNA core encapsulation with over 80%encapsulation efficiency.In addition,it showed classical morphology and uniform size distribution.In vitro experiments,the mRNA nanovaccine showed superior transfection efficiency in DC and no obvious cytotoxicity was observed in BMDC.The mRNA nanovaccine showed good stability and expression upon freeze-thaw cycle and lyophilization.2.In in vitro experiments,the mRNA nanovaccine could stimulate the elevated expression of CD40,CD80,and CD86,which marked the maturation level was enhanced.In the meanwhile,the MHC I complex of BMDC was increased obviously upon the treatment of the mRNA nanovaccine.According to our results,our mRNA nanovaccine could promote the maturation and antigen-presenting ability of DC,thus activating T cells in the end.In addition,high-level secretion of IL-2 was detected by ELISA in the medium of activated DC with T cells.In the meanwhile,T cell proliferation was also detected by flowcytometry.3.In vivo study,we detected a persistent signal at the injection location over 48 hours after the vaccination.And no body weight change and abnormal behavior were observed in the treatment group.Such results indicate that mRNA nanovaccine has the capability of expression in vivo with good biosafety.4.In vivo study,mRNA nanovaccine was able to induce the maturation of DC subunits in the draining p LN,which was shown as the maturation marker CD86 elevated and reached its peak after 48 hours.In addition,the mRNA nanovaccine promoted the elevation of CD8~+T cell proportion.And IFN-γ~+cells reached a peak on day 5 after immunization.In the experiment of MC38-OVA inhibition,compared with other control groups,mRNA nanovaccine could significantly inhibit tumor growth and increase the concentration of CD8~+T cells in tumor tissue to induce the Th1-biased specific immune response.Apparent IFN-γ~+CD8~+T cells could be tested in SPL,p LN and tumor tissues.Our mRNA vaccine led to the expression of antigen-specific CD8~+T cells.5.In vitro study,mRNA nanovaccine could efficiently induce BMDC to produce the inflammatory cytokines,such as TNF-α,IL-1β,IFN-βand CCL5.To further investigate the mechanism behind,we used the BMDC from STING,TRIF,TLR7,MAVS-KO mice that individual pathways were knock out,respectively.After stimulation by mRNA nanovaccine,the secretion of type I interferon IFN-βwas hindered upon the knocking out STING pathway.However,in TLR7-KO BMDC,IFN-βwas only partially influenced.And the integrity of MAVS pathway was essential for TNF-αrelease.Interestingly,EDOPC in the formulation is crucial for induction of immune response.By replacing EDOPC with DOTAP or DOPC,the secretion of IFN-βand TNF-αwere completely wiped out.Besides,the phosphorylation of crucial marker in downstream of TLR7,NF-κB and STING pathway IRF7,P65,TBK1 and IRF3 were detected in western blot.Here in our study,STING pathway plays an important role in mRNA nanovaccine function,EDOPC is a key factor for stimulating the immune responses and production of cytokines.6.Based on the findings above,we compared the influence of STING and MAVS pathways on mRNA nanovaccine.In contrast with WT group,the DC maturation level,activation of CD4~+and CD8~+T cells were obviously hindered after knocking out the STING pathway.The frequency of IFN-γ~+cell was also lower in STING-KO group.In contrast,no significant changes of antigen-specific CD8~+T cells and CD44~+CD8~+memory T cell proportion were observed in the MAVS-KO group.Finally,the impact of STING pathway on therapeutic effect of mRNA nanovaccine was examined.From the tumor growth curve,the ability of MC38-OVA inhibition was impaired at a certain extent,which indicated that STING pathway was essential for our mRNA nanovaccine.Conclusions:In this study,we designed an mRNA nanovaccine that can effectively deliver the mRNA to DC with adequate self-adjuvant effect.This mRNA nanovaccine can inhibit the tumor growth in murine MC38-OVA colon model by generating antigen-specific tumor cells elimination.The importance of STING pathway on mRNA nanovaccine was elucidated.By adjusting the release of IFN-β,it has obvious influence on the DC antigen presentation and T cells stimulation.In the meanwhile,cationic lipid EDOPC in our formulation is the key factor of STING pathway of inflammatory immune response.