Preparation And Application Of MUC1 MRNA Tumor Nanovaccin

Due to factors such as aging population,environmental pollution and lifestyle,the incidence of tumors is continuously increasing and has become one of the serious causes threatening human health and life.In recent years,the rise of mRNA nanovaccines has provided a new direction for immunotherapy of tumors.Compared with traditional chemotherapy,mRNA nanovaccines have advantages such as high efficiency,specificity and safety.It can not only induce the activation and proliferation of immune cells such as CD4+T cells,CD8+T cells,NK cells,etc.,but also targeted specifically kill tumor cells through T cell-mediated cytotoxicity,thereby enhancing the effect of tumor immunotherapy.However,due to the extreme instability of mRNA,it is easy to be degraded by nucleases both in vivo and in vitro,so it is necessary to use an effective delivery system to transport it into the cells.Currently,the commercialized delivery of mRNA vaccines is mainly based on lipid nanoparticles(LNP),which can improve the delivery efficiency of mRNA.However,due to the complexity of preparation and the need for strict storage conditions,its widespread application is limited to some extent.In this study,we plan to select the MUC1-VNTR gene sequence as a tumor-associated antigen,and combine it with the dual adjuvant self-delivery nanosystem developed by our research group to prepare an efficient mRNA tumor nanovaccine(mMUC1 NPs),providing a new method for the treatment of pancreatic ductal adenocarcinoma.We designed and amplified plasmid DNA containing the MUC1 gene,synthesized mRNA by in vitro transcription,and used adjuvant monophosphatidyl A(MPLA)and CpG oligonucleotides(CpG ODN)as carrier materials to package mRNA into nanovaccines by self-assembly technology.We first studied its particle size,zeta potential,surface morphology,encapsulation efficiency and in vitro stability.In vitro experiments mainly explored the cytotoxicity of mMUC1 NPs and the ability to induce dendritic cells(DCs)uptake,maturation and activation,and the expression time of mMUC1 NPs in vivo was analyzed by immunoblotting.In vivo experiment,using MUC1-positive(MUC1+)pancreatic ductal adenocarcinoma in mice as a tumor model,we evaluated the anti-tumor effect of the nanovaccine and further explored its anti-tumor immune mechanism by analyzing the immune status of mice systemically and locally.The results of characterization proved that we successfully synthesized mRNA by in vitro transcription and effectively loaded it into dual adjuvant self-delivery spherical nanoscale particles.The size of the nanovaccine was uniform(160.53±3.90 nm)and well dispersed(0.20±0.009),stable for more than 30 days in aqueous solution.In vitro cell experiments showed that mMUC1 NPs had no obvious cytotoxicity,and was easily taken up by DCs and translated into proteins.After cocultivation with DCs derived from mouse bone marrow,mMUC1 NPs significantly stimulated the expression of the main stimulatory molecule MHC-Ⅱ and co-stimulatory molecules CD40,CD80 and CD86,and stimulated the secretion of cytokines such as IFN-y and TNF-α.Surface mMUC1 NPs promoted the maturation and activation of DCs,initiating the immune response.In addition,although different storage conditions had little effect on mMUC1 NPs,4℃ was the most effective for long-term storage.The results of in vivo anti-tumor experiments showed that compared with the first-line treatment plan currently used clinically,mMUC1 NPs could significantly inhibit tumor growth in mice with tumors and prolong their survival.Moreover,both the nano vaccines combined with chemotherapy drugs or PD-1 antibodies could exert immune synergy,especially when combined with PD-1 antibodies,the tumor inhibition effect was more significant,and about 30%of the mice achieved complete regression of the tumor,while 83%of mice achieved long-term tumor-bearing survival.In the study of immune mechanism,mMUC1 NPs or the combined use with chemotherapy drugs or PD-1 antibodies could significantly stimulate the proliferation and activation of CD4+T cells and CD8+T cells in lymph nodes,spleen and tumor of mice with tumors,and downregulated the proportion of Tregs in spleen and tumor.At the same time,the combination of mMUC1 NPs and PD-1 antibodies could promote the proliferation of NK cells in the tumor microenvironment and secrete chemokine XCL1 to recruit DCs,thus increasing the proportion of lymphocyte infiltration in the tumor microenvironment and achieving specific killing of tumors.In addition,the combination of mMUC1 NPs and chemotherapy drugs or PD-1 antibodies could promote the secretion of cytokines IL-12 and IFN-y,induce the proliferation of memory CD4+T cells,achieve long-term immune memory and effectively prevent tumor metastasis and recurrence.In conclusion,the use of dual adjuvant self-delivery carrier-loaded tumor antigenspecific mRNA to construct mRNA tumor nano vaccines can stimulate the body to produce highly efficient anti-tumor immune responses,and is a promising anti-tumor immune approach with broad application prospects.