Investigating The Effects Of RAAV₂ Mediated ApoAI And SR-BI Double Gene On AS

Atherosclerosis (AS), a kind of general arterial disease, is a leading dangerous disease over the world with high morbidity and mortality. The etiopathogenisis of this disease, however, need to be further determined, which leads to unsatisfactory curative effect.Therefore, the study of its etiopathogenisis and more effective treatment is very significant. Some research showed that gene therapy had extensive perspective and offered a promising new approach for the treatment of AS. In our study, we determined the feasibility of simultaneous over-expression of apoAI and SR-BI by AAV-mediated gene transfer and our results showed that it had a favorable effect on diet-induced hypercholesterolemia and arterioscl-erosis in rats.Methods and Results:1. The method of packaging and purify rAAV-apoAI-IRES-SR-BI.The AAV helper-free System was used as basis to generate recombinant AAV. The IRES sequence was amplified by PCR using pAAV2-IRES-hrGFP vector as template, and subcloned into PMD18-T Vector, APOAI and SR-BI gene was amplified by PCR using human fetal liver cDNA as template.and also subcloned into PMD18-T Vector. First of all. The apoAI sequence of plasmid PMD18-T-APOA1 was cut down and subcloned into pAAV2-IRES-hrGFP vector multiple clone sites (between restriction sites BamHI and EcoRI).When the pAAV2-APOAl IRES-hrGFP was Successfully Constructed. Subsequently The IRES and SR-BI sequences of PMD18-T-IRES and PMD18-T-SR-BI were cut down respectively. The two fragments were fusion utilizing T4 DNA ligase through their restriction sites Mlul. Meanwhile pAAV2-APOAl- IRES- hrGFP was digested with Sall and BglⅡ. The IRES-hrGFP were cut off and replaced by IRES-SR-BI.Then, recombinant plasmid pAAV2-APOA1-IRES-SR-BI, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete pAAV2-apoAI-IRES-SR-BI packaging. The GFP labeled pAAV2-apoAI-IRES-hrGFP was simultaneously packaged by using the parallel plasmid. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293cells.They were purified with the method single-step column purification (SSCP).The high purity of rAAV was determined by Western-blotting, The virus titer and vitality was measured by dot-blot and infecting HepG2 cells, The titer of rAAV-apoAI-IRES-hrGFP was 6.716×1010v.g/ml, and The titer of rAAV-apoAI-IRES-SR-BI was 1.2376×1011v.g/ml.3. rAAV2-apoAI-IRES-SR-BI mediated gene transfer and expression in HepG2 Cells, which influenced the function of RCT.We measured the transduction efficiency of HepG2 cells infected with 1×102-1×106 rAAV2-apoAI-IRES-hrGFP particles per cell.GFP expression in AAV-HepG2 cells could be observed under fluorescent microscope 72h after transfection.To detect the apoAI and SR-BI mRNA and protein expression levels in HepG2 cells which either mock infected or infected with the rAAV2-IRES-hrGFP,rAAV2-apoAI-IRES-hrGFP and rAAV2-apoAI-IRES-SR-BI three vectors respectively three days later, the apoAI and SR-BI mRNA and protein expression was respectively assessed by means of RT-PCR and western blotting.HepG2 Cells were seeded at a density of 1×105 cells/well in a 12-well tissue culture plate and cultured for 24 h to 60-80% confluence.HepG2 Cells were either mock infected or infected with the rAAV2-hrGFP, rAAV2-apoAI-IRES-hrGFP and rAAV2-apoAI-IRES-SR-BI three vectors respectively for 2 h at 37℃at 5×104 particles per cell. Two hours later the transfection medium was removed, and fresh complete growth medium was added. Forty-eight hours post-transfection, Then they were observed under the inverted fluorescent microscope. Low density lipoprotein (LDL) was isolated from human plasma by one time density gradient centrifugation, and Cu2+ at a concentration of 10μmol/L was used to induce the oxidation of LDL (ox-LDL).after 48h later, HepG2 cells were co-incubated with 50μg/ml ox-LDL, respectively, Cholesterol net content was determined by high performance liquid chromatography analysis after the next 24 hours later. The results suggested that there was a dose-dependence between the AAV vector particles per cell and transduction efficiency within a certain range.When rAAV vectors were used at concentration of 5×104 per cell, transduction efficiency was more than 70%. The result also suggested that exogenous apoAI mRNA and protein level was strongly expressed in rAAV2-apoAI-IRES-hrGFP and rAAV2-apoAI-IRES-SR-BI transduced HepG2 cells,but the rAAV2-IRES-hrGFP transduced HepG2 cells and No transduced expression of endogenous apoAI was very low. Total cholesterol content of HepG2 cells was decreased in rAAV2-apoAI/SR-BI group compared with rAAV2-GFP group, rAAV2-apoAI group and control group. The ratio of CE/TC in rAAV-apoAI group and rAAV2-apoAI/SR-BI group was increased compared with rAAV2-GFP group control group. Our results indicated that rAAV2-apoAI/SR-BI group and rAAV2-apoAI group decreased cellular cholesterol content in HepG2 cells. And the rAAV2-apoAI/SR-BI group is stronger than rAAV2-apoAI group on decreasing cellular cholesterol content in HepG2 cells.4. The establishment of AS rat model and comparison of the effects among rAAV2-GFP,rAAV2-apoAI and rAAV2-apoAI/SR-BI on AS in VIVO.Thirty-two male healthy SD rats were randomly divided into four groups. The control group rats were fed with basic food while in other three groups was loaded with high fat diet for 3 months. The levels of plasma total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were determined with biochemical techniques before and after the establishment of AS rat model at the third month. The ultrasonic changes in the aorta were determined after 3 months. The results showed that rats were fed with high fat diet food were observed in the atherosclerotic plaque presented in the aorta accompanied with the obvious increase of serum lipid after 3 months.Four experimental groups were administered the following AAV vector constructs:rAAV2-apoAI-IRES-SR-BI, rAAV2-apoAI-GFP, rAAV2-IRES-GFP, and PBS. ApoAI and SR-BI gene expression was detected using RT-PCR.The apoAI and SR-BI protein expression was determined by Western-blotting and ELASA. Diet-induced hypercholesterolemia and Atherosclerosis in rats'model was adopted and rAAV2 was administered through the tail vein injection. GFP expression could be detected at eight weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, Plasma total and LDL-cholesterol concentrations were significantly reduced (p<0.05) compared to rAAV2-IRES-GFP (rAAV2-GFP) treated group. No effect on HDL-cholesterol concentrations occurred. Ultrasound determined intima-media thickness also has been significantly reduced compared to rAAV2-GFP treated group. Serum hs-CRP and SOD levels increased significantly (p<0.01). Serum MDA levels decreased significantly. We detected gene mRNA expression in atherosclerosis rats'model. These gene included NF-KBp65, ICAM-1, VCAM-1, IL-1β, MCP-1, MMP-2,MMP-9 IKBα, eNOS.Conclusion:We used rAAV as a gene transduction system by successfully inserting the apoAI and SR-BI genes in this vector, allowing them to be efficiently and stably co-expressed. The apoAI and SR-BI proteins that were expressed from the rAAV2-apoAI-IRES-SR-BI vector enhanced reverse cholesterol transport in vitro and in vivo. Our experiments also establish a foundation for investigating the synergistic biological effects of apoAI and SR-BI in vitro and in vivo and provide theoretical support for gene therapy of diet-induced hypercholesterolemia and Atherosclerosis with our recombinant virus.