Production And Purification Of Myocardium-Specific Recombinant Adeno-Associated Virus Vector Carrying VEGF165 Gene

Background and ObjectivesNowadays,the clinical treatment of ischemic heart disease?IHD?is mainly conventional drug therapy and revascularization therapy?percutaneous coronary intervention,coronary artery bypass grafting?.However,even patients who receive medication or revascularization treatment will still have symptoms such as repeated angina or heart failure,and gene therapy shows potential in increasing collateral vessel growth and reducing myocardial ischemia,becoming the hope of IHD treatment.Vascular endothelial growth factor?VEGF165?has been proved to play a role in anti-cardiomyocyte apoptosis,inducing stem cells to participate in the repair of ischemic myocardium and improving the stability of ECG after myocardial infarction.Therefore,we selected VEGF165 as the target gene for IHD gene therapy in this study.In the selection of gene vectors,adeno-associated virus?AAV?,because of its low immunogenicity,high safety,good tissue specificity,wide host range,and the ability to induce long-term stable expression of foreign genes,has surpassed other viruses and become the most promising gene therapy vector for IHD.In this study,we selected the AAV9 with myocardial tropism,combined with the myocardial-specific c Tn T promoter to mediate the long-term stable expression of the VEGF165 gene in myocardium,so as to exert a lasting and effective myocardial protection.However,the large-scale packaging and purification of rAAV is a key issue that restricts the rapid development of rAAV drugs.and there are many problems such as high production cost,complicated operation,poor purity of the finished product,low recovery rate,and more empty shell viruses.Therefore,this study aims to construct a myocardium-specific recombinant adeno-associated viral vector?rAAV9-c Tn T-VEGF165-e GFP?carrying VEGF165 gene for IHD gene therapy,and explore a simple and effective method for the preparation and purification of rAAVMaterials and MethodsThe research methods of this study mainly include:1)Construct the recombinant vector plasmid p AAV-c Tn T-VEGF165-e GFP by genetic engineering,and verify the DNA sequence of VEGF165 gene on the vector by colony PCR and gene sequencing,and then verify the expression of VEGF165 gene by Western Blots?WB?;2)We extracted sufficient amount of three plasmids?p AAV-c Tn T-VEGF165-e GFP,p AAV-R₂C₉,p Helper?,and used plasmid co-transfection method to package rAAV,using fluorescence microscope to evaluate the virus packaging efficiency,and verify the packaged rAAV virus by WB;3)The cation exchange chromatography method was used to purify the virus solution after harvesting,including the first round of purification at higher p H and the second round of further purification characterized by strict intensive gradient elution.The purified product was analyzed by SDS-PAGE and Western blot?WB?,and the virus titer was determined by Q-PCR method.Finally,the virus particles were further verified and analyzed by transmission electron microscopy.Results1)The results of colony PCR and gene sequencing indicated that the recombinant plasmid p AAV-c Tn T-VEGF165-e GFP was successfully constructed and the inserted VEGF165 gene sequence was correct;WB results proved that VEGF165 can be normally expressed;2)The results of fluorescence microscopy showed that the packaging efficiency of the virus was high,up to 70%-80%;3)SDS-PAGE results showed that the virus purification effect was significant.The first round of purification removed a lot of protein impurities,and the second round of purification also significantly removed some impurities.The purified product was confirmed to be rAAV virus by WB and electron microscopy.The titer of the virus was2.15×10¹²GC/m L measured by Q-PCR method,and the electron microscopy results further revealed that the empty particles contained in the final virus production were very few,and the percentage of genome containing vectors can reach 99%.ConclusionsIn this study,we successfully prepared a myocardium-specific rAAV gene therapy vector?rAAV9-c Tn T-VEGF165-e GFP?carrying the VEGF165 gene,and developed a simple and effective purification method for scaled rAAV based on cation exchange chromatography.