Screen Of Traditional Chinese Medicine Components That Enhance Recombinant Adeno-associated Virus Transduction And Mechanism Identification

Adeno-associated virus(AAV)is a non-pathogenic,small non-enveloped DNA virus and belongs to the parvoviridae.Due to its replication-defective property,a helper DNA virus is required to complete its replication cycle.The recombinant adenoassociated virus(rAAV)vector has emerged as one of the most commonly used and promising vectors.So far,rAAV has been used for clinical gene therapy for numerous diseases,including Hemophilia,Leber congenital amaurosis,Parkinson's disease and heart failure.Compared with other gene therapeutic vectors,rAAV possesses a lot of advantages such as a good safety profile high stability,low pathogenicity,weak immunogenicity,broad host cell range,long term gene expression and so on.The world's first gene therapy drug is based on rAAV1.Nevertheless,due to the limited transduction efficiency,the rAAV-mediated treatment is still highly costed.In addition,the high does of rAAV will induce host immune response.Interaction with cellular stress pathways plays a key role in motivating the life cycle of many kinds of latent virus.As previous studies have demonstrated that cellular stressors frequently increase the transduction of rAAV vectors and may be even a substitute for helper virus.In consideration of the safety,minimal side effect and good adaptability of the TCM active components,we choose them as stressors for research.At present,some active components have already became a member of the family which enhance rAAV transduction efficiency as proteinase inhibitors,such as Celastrol,pristimerin and so on.As a kind of TCM medicines,Salvia miltiorrhiza bunge has a long history in China.As previous studies have demonstrated that Salvia miltiorrhiza Bge has known effects on HBV and HIV life cycles.Therefore,this project selected active components which can enhance the transduction efficiency of rAAV from Salvia miltiorrhiza bung first.In this study,a integrated strategy that combines the transduction assay in vitro and the immunofluorescence assay has been developed.To verify the feasibility of this strategy,Salvia miltiorrhiza bunge was selected as a target drug for active components screening.Firstly,we successfully produced various types of rAAV vectors that can be used for subsequent experiments.Secondly,we improved experimental methods and strategies based on previous experiments and have successfullyacquire two active components,salvianolic acid B(Sal B)and Salvianic aid A from Salvia miltiorrhiza bunge.Then the activity of Sal B was verified by experiments such as cell viability,drug efficacy relationship,time-effect relationship,stability exploration,and universality.The results showed that Sal B does have the ability to promote the transduction efficiency of rAAV in HEK293 cells.Additionally,it is suitable for other cell lines.Therefore,we used immunofluorescence technique to locate the distribution of rAAV vector particles within cells to late endosomal the target of Sal B.Experiments confirmed the role of Sal B as a target.In conclusion,through the above strategies,we successfully selected two active ingredients from Salvia miltiorrhiza and identified the target of Sal B through cell imaging technology,and elucidated the mechanism of sal B to improve the transduction of rAAV.