Design And Application Of Nucleic Acid-based Cancer Vaccine And Pyroptosis Strategies For Cancer Treatment

Although there have been tremendous advances in the clinical diagnosis and treatment.cancer remains to be one of the most lethal diseases in the world due to its high risk of recurrence and high heterogeneity.Cancer immunotherapies such as immune checkpoint blockade therapy,CAR-T cells therapy and cancer vaccines have entered clinical use,but the overall response rate still needs to be improved.Therefore,new strategies for cancer treatment need to be explored.In the development of novel cancer therapeutic strategies based on nucleic acids,mRNAs encoding cancer antigens or therapeutic proteins show inspiring promise due to their high biosafety and ability to rapidly re-express proteins.However,the practical application of mRNAs is still limited by restricted half-time,inherent immunogenicity,as well as poor stability.To overcome the above problems,this thesis firstly constructed an in vitro transcription platform for autonomous design and synthesis of mRNA to improve expression efficiency and stability,as well as reduce immunogenicity.Specifically,sequence design of mRNA on template before transcription,including "NASAR" UTRs and segmented poly(A)tail;doping pseudouridine(m1Ψ)during transcription,as well as Cap 1 modification of mRNA after transcription.On the basis,an mRNA-based cancer vaccine strategy was developed using fluoroalkane-grafted polyethyleneimine(F-PEI)as the carrier.F-PEI/mRNAOVA was confirmed to enhance pattern antigen OVA presentation via MHC class I molecules and activate specific CD8+T cell immune response.Then,a personalized mRNA cancer vaccine based on mRNAneoantigen,an mRNA molecule encoding multiple epitopes of neoantigens,was designed for MC38 colon cancer model.As the therapeutic vaccine,F-PEI/mRNAneoantigen could significantly inhibit tumor growth and prolong the survival time of tumor-bearing mice.Based on the design of nucleic acids described above,pyroptosis with strong inflammation for immunosuppressive tumor therapy was exploited to design a treatment strategy,which is based on the N-terminal domain of gasdermin D(GSDMD-NT).Bypassing the endogenous regulatory mechanism of pyroptosis,production of GSDMD-NT in cells could directly induce pyroptosis through intracellular delivery of encoding plasmid or mRNA.Firstly,we demonstrated that intracellular expression of Flag-GSDNT induced tumor cells pyroptosis using the encoding plasmid(pFlagGSDNT)and bioreducible branched poly(β-amino ester)s(BPAE-SS)as the carrier at cellular level.In the therapeutic experiment of colon cancer model,intra-tumoral injection of BPAE-SS/pFlag-GSDNT(wt)significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice,achieving a survival rate of 40%,thus validating the feasibility of the pyroptosis strategy in cancer treatment.Subsequently,mRNA encoding Flag-GSDNT was constructed and synthesized,which can efficiently induce pyroptosis in vitro.Despite the fact that the treatment efficacy of mRNAFlag-GSDNT in the breast cancer model was limited by the in vivo delivery efficiency,it has laid a solid foundation for the following development of mRNA-based pyroptosis strategy.In conclusion,on the basis of nucleic acid molecules’ design,this thesis constructed two novel cancer therapeutic strategies:mRNA cancer vaccine and intracellular expression of GSDMD-NT inducing pyroptosis.The feasibility of the strategies and their application value in cancer therapy were firmly demonstrated.