Novel Adeno-associated Virus Vector And Applied Research

Adeno-associated viral vectors have been widely used for gene therapy in animal models as well as human clinical trials. Recent studies using vectors derived from alternative AAV serotypes or modified capsids have shown improved potency and broadened application range of AAV vectors.In the first part of this study, we set up an AAV vector production system by using recombinant herpes simplex virus carrying rep and cap gene as helper.6 types of AAV vectors (AAV1,AAV2,AAV5/5,AAV2/5m,AAV2/8m,AAV8) were produced and the titer was determined by digoxin labeled dot blot, and capsid proteins of each AAV virus were analyzed by SDS-PAGE.In the second part, utilizing the properties of resistance to heating and organic reagents of AAV particles, we established a novel method named "reverse infection" for AAV vectors, by pre-coating quantified AAV vectors on 96-well culture plate and air drying for storage. Different quantity of AAV vector expressing Gaussia luciferase (Gluc) gene added to each well (vg/well) as well as different number of cells added to each well (cells/well) were evaluated for their relationships to detecting sensitivity of the Gluc gene expression. Different coating solutions were screened and selected one for final use that well maintained AAV's infectious activity in dried condition and at various temperatures including 4,37,42,56℃. Then we utilized the method to evaluate the transduction efficiency of 6 types of AAV vectors (AAV1,AAV2,AAV5/5,AAV2/5m,AAV2/8m,AAV8) for 16 cell lines (BHK21,HEK293,BEAS-2BS,C2C12,L02,Huh7,HepG2,Hepa1-6,L/S-39,CT-26,A549,HeLaS3,U937,K562,SP2/0,A20). The results showed the transduction efficiency were high for BHK21,HEK293,U937,BEAS-2BS cells, and low for CT-26,SP2/0,A20 and L/S-39. The transduction efficiency was found various for same cells with AAV2 highest and AAV1 second. For HeLaS3 cell, transduction efficiency of AAV1 was higher than AAV2. Adding sodium butyrate could significantly increase the expression mediated by AAV vectors, especially for AAV2, and then AAV1. The in vitro transduction efficiency of AAV8 vector was much lower than AAV2 and AAV1 in all the cells detected.As shown above, rAAV could transduce cells effectively in a reverse infection way, showing a great advantage for using AAV vector as carrier for biosensor. Then we tried to detect microRNAs activity profiles by using our AAV RI Array method. The miRNA sensor carried by rAAV2 was named as Asensor. Asensor contained a Gaussia luciferase (Gluc) gene expression cassette with one copy of miRNA-complementary sequence in its 3'UTR and a firefly luciferase expression cassette acting as an internal control for calibrating transduction titers. Asensors carrying various miRNA-complementary sequences of equal transduction titer were separately coated on 96-well cell culture plate, and dried. The Asensor without any miRNA-complementary sequence was coated as control. Different Asensors were arranged orderly in the culture plate resulting in'Asensors array'. Cells to be detected were added with equal number to wells of the Asensor array plate. The Gluc activities in the supernatant were detected after 24-48h. The relative microRNA activities could be presented by comparing Gluc activity of the control Asensor to that of the specific miRNA Asensor. This study provides a novel strategy for detecting miRNA activity profiles.In the third part of this study, we tried to establish chronic HBV mouse model using AAV8 vector. AAV8 vector was reported having an excellent in vivo transduction efficiency and liver tropism.A recombinant AAV8 virus (rAAV8-HBV1.3) carrying 1.3 copies of HBV (ayw subtype) genome DNA was prepared with titer of 1×10e12 vg/mL. At first, a group of C57BL/6 mice (n=3) were tail injected with 200μL of rAAV8-HBV1.3 for each. HBsAg and HBeAg in sera(1:4 diluted) were detected by HBV ELISA kits. The HBsAg and HBeAg were found positive and lasted for at lest 10 weeks. Among them, HBsAg levels showed an'increasing-decreasing-increasing'change (lowest at the 4th week), while HBeAg levels kept high and relatively stable. The three mice were sacrificed after 10 weeks and blood and liver tissue were taken for assay. HBV S gene fragments with length of 300bp were detected positive by PCR for liver genome DNA samples from all three mice. Then the HBV DNA copies in liver tissue genome DNA samples and in sera was quantified with real-time fluorescence quantitative PCR kit. Results showed HBV DNA in sera were 4.2×103,3.6x103,2.5×103copies/mL, and in liver 8.0×106,5.7×106,2.6×106 copies/g liver tissue. In order to realize the replication of the HBV DNA in liver infected by rAAV8-HBV1.3, a pair of primers was designed at the two ends of the non-repeat region in HBV1.3 DNA towards outside to find if the HBV genome could cycled and in HBV style or in AAV style. Positive PCR products were found in all three liver DNA samples, and their length suggested that cycled HBV DNA were formed. At last, we compared primarily the responses of different species of mice (Balb/c and C57BL/6 mice, n=3 for each) to rAAV8-HBV1.3. Results showed that the three C57BL/6 mice and the three Balb/c mice were all HBeAg-positive (1:4 diluted) from the first week on, and maintained at high and relatively stable levels thereafter. For Balb/c mice, the HBsAg level increased promptly within 2 week after injection and then dropped to levels below the cutoff value. The HBsAg levels were persistently positive for 2 C57BL/6 mice while transiently positive for the other one. Further experiments will include more number of mice and investigate longer time to study the characteristics of our nontransgenic mouse model for HBV persistence. Our work will provide opportunities to investigate the mechanisms of HBV persistence.