Development And Application Of NF-?B-based Gene Expression Manipulation Techniqes

NF-?B is a transcription factor which is binding to specific DNA sequences.In the unactivated state,NF-?B binds to I?B,its inhibitory protein,and stays in the cytoplasm.The inhibitory protein I?B could be degraded when cells are stimulated,such as virus infection,ultraviolet radiation,cytokines(TNF?,etc.),by the intracellular NF-?B signaling pathway.Then,NF-?B enters the nucleus,and binds to nuclear DNA targets,controlling the expression of target genes,thereby participating in the physiological and pathological processes such as cells growth,division,apoptosis,renewal,inflammation,and canceration.NF-?B is activated in inflammation and cancer,and has become an important target for drug screening and disease treatment,such as NF-?B inhibitors.However,NF-?B is a double-edged sword,traditional NF-?B inhibitors have not become clinical drugs because of the significant side effects.Therefore,the exploration of new research strategies are required for the value of NF-?B-based biomedical applications.Three novel gene expression control technologies based on NF-?B has been developed,and their biomedical application value has been explored in this study.1.Development and Application of NF-?B-based Highly Active Eukaryotic PromotersHuman cytomegalovirus(hCMV)major immediately early promoter(MIEP)is the most active eukaryotic promoter which has been widely used in basic research of life sciences and various types of genetic engineering,gene therapy,and gene editing.In this study,the high-affinity artificial sequences has been screened by the SELEX system,and has been used to replace the natural NF-?B binding sites(?Bs)in human MIEP.Then,various mutant MIEPs(mMIEPs)has been constructed.Three mMIEPs with significantly higher transcriptional activity than wild-type MIEP have been screened,by the detection of various transcriptional activity evaluation systems.These mMIEPs can be widely used as artificial promoters in the fields of genetic engineering drug production and gene therapy.In addition,a new method for the rapid construction and screening of various active promoters have been provided,which is useful for promoter engineering.And a series of mMIEPs with multiple transcriptional activities have been preparaed,which can be used for flexible control of the expression level of target genes in synthetic biology.In this study,the modified artificial promoters have been applied to secretory expression of hG-CSF protein.The study on the expression of this protein not only can deeply verify the expression efficiency of the promoters,but also provides an important basis for the application of mMIEP promoters in the genetic engineering pharmaceutical.2.Development and Application of Novel NF-?B Activity-Suppressing Gene VectorsIt has been testifided that NF-?B plays an important role in tumor cell proliferation,angiogenesis,invasion and metastasis by regulating its target genes.Moreover,the expression of NF-?B family proteins in tumor tissue cells is significantly enhanced compared with normal cells.NF-?B has become an important target for new drug treatment and research.Therefore,many different NF-?B inhibitors have been developed.But,NF-?B is not only a disease-associated transcription factor,it also plays an important role in the normal physiological functions of cells,and its excessive inhibition will produce serious side effects.Accordingly,these inhibitors are difficult to become a commercially clinical drug.In order to overcome this limitation,the classic NF-?B specific decoy sequence has been used as a NF-?B-specific promoter element to fuse with the minimal promoter sequence,constructing a NF-?B regulation decoy minimal promoter(DMP).In this study,a novel NF-?B activity-controlled gene vector have been constructed based on this promoter.These vectors can express artificial miRNA targeting NF-?B RelA,under the control of the DMP promoter.It has been confirmed that,the vector can sense and regulate the activity of intracellular NF-?B.Expression of the vector forms a perfect feedback loop to achieve self-regulation of NF-?B.The higher the activity of NF-?B,the higher the transcriptional activity of the DMP promoter and the higher the expression level of amiRNA.The DMP-amiRNA system can moderately inhibit NF-?B activity in NF-?B over-activated cells(e.g.tumor cells),causing apoptosis of tumor cells,but does not have a significant effect on normal cells.In this study,a novel NF-?B activity inhibitor DMP-amiR533 has been obtained.A new strategy to inhibit the activity of NF-?B has been acquired.The selected DMP-amiR533 has important application value in the treatment of NF-?B hyperactivated diseases such as inflammation and cancer.3.Development and Application of NF-?B-activated Tumor cell-specific GeneExpression VectorsIn recent years,immunotherapy has contributed greatly to cancer treatment.However,the cancer immunotherapy is still prevented by tumor cell-specific antigens.Therefore,researchers are looking for new antigens by tumor sequencing and bioinformatics prediction,to develop personalized cancer immunotherapy.However,this strategy has high technical complexity,high cost,and long cycle.In this study,we have developed a novel immunotherapy by developing tumor cell-specific gene expression technology.Specific proteins or polypeptides can be expressed on the surface of tumor cells by NF-?B-specific gene expression vectors.These proteins or peptides can be used as artificial new antigens,to distinguish tumor cells from normal cells and stimulate the immune system to kill tumor cells.In this study,a NF-?B-activating gene expression(Nage)vector system has been constructed and validated.This vector uses the DMP promoter to enable the downstream effector genes specifically expresse on various tumor cell surfaces.The proteins or peptides include HBsAg,CRT,and SBP.Then,the Nage vector has been packaged in adeno-associated virus(AAV)as a viral vector drug for tumor immunotherapy.The final result has showed that recombinant AAV can significantly inhibit tumor growth in vivo,and there is no toxic side effects.The novel cancer gene immunotherapy proposed in this study has important application value in the field of cancer treatment.In summary,a novel gene expression control technology based on the transcription factor NF-?B has been developed in this study.A novel promoter(mMIEP)with significantly improved transcriptional activity,a novel NF-?B activity control vector(DMP-amiR533),and NF-?B-activating gene expression(Nage)vectors have been developed.These new NF-?B-based gene expression control technologies have important application value in the field of biopharmaceuticals and gene therapy.