Construction And Identification Of Recombinant Adeno-associated Virus Vector With Overexpressing VEGF165 Regulated By Cardiac-specific Promoter And Two-step Transcriptional Amplification System

Background and Objectives At present,with the continuous development of the economy and society,the incidence of cardiovascular diseases is increasing year by year.Acute myocardial infarction is one of the most serious cardiovascular diseases.Common treatments for myocardial infarction include drugs,thrombolysis,percutaneous coronary intervention(PCI),but most patients still miss the best treatment period.The discovery of VEGF165 provides a new direction and method for the treatment of ischemic heart disease such as acute myocardial infarction.Many studies have found that VEGF165 protein can promote angiogenesis,increase vascular permeability,inhibit cardiomyocyte apoptosis and ventricular remodeling.However,the half-life of VEGF165 protein is short,and it decomposes quickly in vivo.So the clinical application of VEGF165 protein therapy is limited.Gene therapy has become a promising treatment for ischemic heart disease.Adeno-associated virus(AAV)vector has become the most commonly used vector in gene therapy because of its high safety,long duration of gene expression,and low immune response to the host.In this study,VEGF165 was selected as an important target gene for gene therapy of ischemic heart disease,which was transcribed and expressed under the regulation of cardiac troponin T(cTNT)promoter,and then to construct the adeno-associated virus vector:p AAV-cTNT-VEGF165-Flag-EGFP.The cTNT promoter not only avoids the side effects of VEGF165 regulated by CMV,a broad-spectrum promoter from Cytomegalovirus,on other normal tissues and organs,but also has higher gene expression efficiency than other myocardial-specific promoters.Previous studies have found that although cTNT promoter is highly expressed than other cardiac specific promoters,the expression of target genes is still low.Therefore,on this basis,the gene elements of two-step transcription amplification(TSTA)systems were introduced to enhance the gene expression regulated by cTNT promoter.In this study,we constructed recombinant plasmid p AAV-cTNT-TSTA-VEGF165-Flag-EGFP by genetic engineering,and then to verify the target gene expression rate of the recombinant plasmid carrying TSTA system,production and purification of recombinant adeno-associated viruses.It provides a basis for the further study of the protective and therapeutic effects of recombinant adeno-associated virus-carrying VEGF165 gene on ischemic heart disease and promotes the clinical transformation of VEGF165 gene therapy.Methods(1)Access and synthesis the sequence of TSTA,PCR amplification and detection sequence is correct.(2)We connect the TSTA with the previously constructed vector plasmid p AAVcTNT-VEGF165-Flag-EGFP for homologous recombination,and then perform colony PCR and sequencing on the ligation product to detect the connection result of the recombinant vector p AAV-cTNT-TSTA-VEGF165-Flag-EGFP.(3)Western blot was used to detect the expression of recombinant vector p AAVcTNT-TSTA-VEGF165-Flag-EGFP in HEK-293 T cells.(4)The GFP fluorescence expression levels of recombinant plasmid p AAV-cTNTTSTA-VEGF165-Flag-EGFP and pre-vector plasmid p AAV-cTNT-VEGF165-Flag-EGFP transfected HEK-293 T were detected by flow cytometry.(5)AAV was packaged by co-transfection with three plasmids,and the packaging effect of AAV was estimated by observing the fluorescence expression level.The AAV capsid protein was verified by Western Blot to determine the packaging success.(6)The recombinant AAV was purified and concentrated.The genome copy(GC)of recombinant AAV purified and concentrated was determined by fluorescence quantitative PCR(Q-PCR).Finally,the gene expression of recombinant AAV was verified by infected cells.Results(1)Sequencing results showed that the TSTA sequence was correct.(2)Colony PCR and sequencing results showed that the recombinant plasmid p AAVcTNT-TSTA-VEGF165-Flag-EGFP was constructed successfully.(3)Western Blot detection showed that the recombinant vector plasmid p AAVcTNT-TSTA-VEGF165-Flag-EGFP could stably express the target protein.(4)Flow cytometry analysis showed that the GFP expression efficiency of p AAVcTNT-TSTA-VEGF165-Flag-EGFP plasmid was at least 50 times higher than that of p AAV-cTNT-VEGF165-Flag-EGFP plasmid.(5)The fluorescence microscope observation,AAV packaging effect was good.Western Blot results showed that r AAV capsid protein was correct and r AAV packaging was successful.(6)After the Q-PCR results were transformed and calculated,the virus titer was 2.74×1012 GC/ml.The infected cells proved that the cTNT promoter had myocardial cell specificity,and the expression of target protein of r AAV carrying TSTA system was significantly higher than that of r AAV without TSTA system.Conclusions In this study,we successfully constructed a gene therapy vector p AAV-cTNTTSTA-VEGF165-Flag-EGFP carrying cardiac specific promoter cTNT,which can express VEGF165 efficiently.cTNT promoter transcriptional activity increased by at least 50 times.And we successfully produced and purified the recombinant adeno-associated virus type9,the final titer can reach the level of animal injection.The target gene was successfully expressed after the infection of myocardial cells.