In Vitro Selection Of Functional Unnatural Nucleic Acids Targeting CTLA-4 And Application Of Tumor Immunotherapy

The human immune system can recognize and clear tumor cells.However,during the development of tumor cells,they can use negative regulatory pathways to recruit immunosuppressive cells,suppress the function of effector T cells,and evade the attack of the immune system.Immune checkpoints are molecules that produce costimulatory or inhibitory signals in the immune response.Among them,cytotoxic T-lymphocyteassociated protein 4(CTLA-4)has been under intensive research.CTLA-4 is a transmembrane protein that exists on the surface of T cells and serves to transmit inhibitory signals.It can inhibit the initiation of T cell immune responses and avoid excessive activation of effector T cells during the body's normal immune process.Because effector T cells play an important role in the anti-tumor immune response,antitumor immunotherapeutics targeting CTLA-4 have also become a research hotspot in recent years.At present,the inhibitors of CTLA-4 mainly include Tremelimumab and Ipilimumab,which are humanized monoclonal antibodies that can effectively block the binding of CTLA-4 and ligands.Both have good clinical efficacy,but use of antibodybased treatments of chronic diseases such as cancer will bring huge cost,manufacturing and regulatory challenges.To make up for the deficiencies of antibodies,researchers recently reported two aptamers that can block the function of CTLA-4.But these aptamer suffer from low affinity and stability.In order to overcome the problems of the above monoclonal antibodies and natural nucleic acid aptamers,this paper proposes to use functional unnatural nucleic acid molecules to inhibit the expression and signal transduction of CTLA-4 at the gene and protein levels.The main work of this paper is divided into the following two aspects: 1)in vitro selection with CTLA-4 protein as the target was performed,and the obtained unnatural nucleic acid aptamers blocked the binding of CTLA-4 and B7 receptor;2)chemically modified deoxyribozymses were designed and synthesized,which could selectively bind and catalyze the cleavage reaction of CTLA-4 m RNA and reduce the expression of CTLA-4 protein.First,in order to obtain unnatural nucleic acid aptamers that target CTLA-4,we established and optimized an in vitro selection method of unnatural nucleic acid aptamers based on capillary electrophoresis.Combining the advantages of capillary electrophoresis technology with high resolution,high screening efficiency and good biological stability of unnatural nucleic acid aptamers,we first generated unnatural nucleic acid libraries by enzyme-catalyzed reactions.In this paper,the conditions of the primer extension reaction,reverse transcription reaction,sample recovery and PCR reaction were optimized,and the established screening method was verified.The murine CTLA-4 protein was used as the target molecule for in vitro selection.After three rounds of selection,an enriched library was obtained,which was sequenced to obtain sequence information of candidate aptamers.Predictive analysis of the secondary structure of candidate aptamers using MFold software shows that the G content is generally high and most of them are stem-loop structures.The dot blot was used to test the affinity of the candidate aptamer to CTLA-4,and the effect of the candidate aptamer to block the binding of CTLA-4 to B7 protein in vitro was tested by ELISA(EnzymeLinked Immunosorbent Assay).Finally,the unnatural aptamer C22T5 with excellent performance is obtained.This work proves that the established capillary electrophoresis SELEX(Systematic evolution of ligands by exponential enrichment)method for unnatural nucleic acid aptamers is reliable and efficient.And unnatural nucleic acid aptamers as CTLA-4 of blockers become possible.In order to reduce the expression of CTLA-4 protein at the gene level,we chose deoxyribozyme 10-23 to cleavage CTLA-4 m RNA.By introducing 2'-methoxy modification to specific sites of deoxyribozyme,the biological stability of 10-23 can be improved.Prolonging the time of 10-23 catalyzing the CTLA-4 m RNA cleavage reaction can improve the cleavage effect.Further,we used multiple 10-23 deoxyribozyme sequences containing the above-mentioned chemical modifications to perform multi-site co-cleavage of murine and human CTLA-4 m RNA under simulated physiological conditions,and found that cleavage efficiency of CTLA-4 m RNA could be improved..In short,through the strategy of chemically modified deoxyribozyme and multi-site co-cleavage,we achieved efficient cleavage of CTLA-4 m RNA in a simulated in vivo environment,laying a foundation for the future application of gene silencing system to cells.In summary,we use unnatural nucleic acid aptamers and chemically modified deoxyribozyme molecules to achieve inhibition of CTLA-4 signaling function at the protein and gene levels,providing new tools and methods for activating immune responses.At the same time,we have established SELEX based on capillary electrophoresis for unnatural nucleic acid aptamers,which provides a new choice for the selection of unnatural nucleic acid aptamers.