Construction Of PR39 Recombinant AAV Secreted Under The HRE Promoter Control And The Effect Of The RAAV On Gene Therapy Of CHD

Object:For studying the effect of PR39 recombinant AAV secreted under the HREpromoter control on gene therapy of ischemia cerebrovascular disease, wesynthesized the minimal HRE, constructed the T-HRE-CMV-PolyA vector, andthen inserted the NT4-TAT-His-PR39 into HRE regulated adeno-associated virusvector, and verified it's expression on the human umbilical vein endothelial cell(HUVEC) line CRL-1730 and it's function of angiogenesis on cadiocyte of pigin hypoxia.Methods:1. Artificially synthesized the minimal HRE/CMV using PCR analysis,cloned the HRE gene by T vector cloning and then assessed it using DNAsequencing.2. Ligate the fragments coded the HRE/CMV into the NT4-6His-PR39with T4DNA ligase, and constructed the pSS-HRE-CMV- NT4-6His-PR39- PolyA- AAV plasmid.3. The HEK-293 cells were co-transfected with three plasmids, and werepackaged into recombinant virus, and the titer of recombinant virus wasdetermined using the dot blot hybridization.4. Equal-volume pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV and emptyvirus (EV) transfected the CRL-1730 cells respectively, and the expression of6×His was examined by immunocytochemistry.5. Eighteen small-pigs were used to establish the models of acutemyocardial infarction (AMI), and then were randomly divided into three parts:experimental group (HRE-AAV-PR39 treated), control group I (physiologicalsaline treated) and control group II (empty virus treated), and the function of theextent of ischemia were assessed by the magnetic resonance (MR). And themyocardial ischemia specimens were collected to observe the differences onmorphology and pathology.ResultsResults:1. According to the records and the database, the HRE was the smallestsequence, and had been synthesized successfully and cloned by inserted into thepGEM-T easy plasmid, and the product conformed to the DNA sequence.2. The HRE had been synthesized massively using single enzyme digestion,and ligated into the modified pSSHGAAV (pSSV9int-/Xba I) vector, and theninserted into the NT4-6His-PR39 fragment after dual enzyme digested by EcoRI and BamH I. The two cloning were both correct, and these results indicatedthat the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV wasconstructed successfully.3. The HEK-293 cells were co-transfected with three plasmids by calciumphosphate precipitation, and were packaged into recombinant virus; the titer of recombinant virus was determined using the dot blot hybridization, and the titerwas 3.4×10~9 p.f.u.4. The expression of 6×His of NT4-TAT-His-PR39 in CRL-1730 cells wasdetected with immunocytochemistry, and the results of the experimental grouphad significance.5. MR perfusion imaging of myocardium showed that the myocardialinfarction size was obviously diminished due to the effect of pSS-HRE-CMVNT4-PR39-PolyA-AAV.ConclusionsConclusions:1. Artificially synthesized HRE can inserted into the upstream of CMVpromoter which is deficient of endo-enzyme recognize site with PCR, and thenthe hypoxia regulated eukaryotic expression vector can be prepared.2. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV can be constructedsuccessfully, and the recombinant virus can be packaged successfully in higherconcentration.3. Results of immunocytochemistry suggest that the HRE in CRL-1730can effectively modulate the expression of downstream geneNT4-TAT-His-PR39 regulated by CMV promoter.4. MR cinematic technique and MR perfusion imaging of myocardium canbe used to evaluate the myocardial infarction size and myocardial perfusionquantitatively and effectively.5. Recombinant pSS-HRE-CMV-NT4-PR39-PolyA-AAV can promote theangiogenesis of myocardium ischemia area, obviously decrease the size ofinfarction and improve the cardiac function.