Basic Research Of Liver Targeting Vector

Objective1. To obtain HBsAg targeting recombinant adeno-associated virus (rAAV) encoding HBsAg-shRNA by molecular biological techniques, and lay a foundation for developing new reagents or drugs for the targeting treatment of HBV.2. To study the biological function of the major subunit variants of human asialoglycoprotein receptor and provide foundation for hepatic targeting therapy through ASGPR.Methods1. In order to construct both expressing shRNA and HBsAg targeting recombinant AAV2 virus,a HBsAg specific peptide was inserted into the 587AA position of AAV2 capside by recombinant PCR and meanwhile a DNA sequence expressing HBsAg-shRNA was inserted into the genome of AAV2.2. The recombinant AAV2 was paked by cotransfected with three plasmids into AAV-293 cells mediated by calcium acid phosphate. The rAAVs (rAAVssy-shHBs) infected HepG2,HepG2215 after purified and quntitated by Real-Time PCR.The efficiency of infection was tested by FACS.3. The specificity of the rAAVssyU6-shRNA-hrGFP on HepG2.215 cell was identified by heparin and HBsAb blocking Test. The silencing effect of the virus on HBsAg, HBeAg gene expression was assessed by ELISA.4. Two major subunit variants of ASGPRH1 (Hla and Hlb) and a minor.subunit variant H2c were cloned from normal human liver tissues. These genes were subcloned into eukaryotic expression vector to construct EGFP fusion protein in order to test their location in cell.5. To constructed a cell line which stably expression ASGPRH1a and H2c. The function of the cell line was tested by FACS.The influence of the split variant ASGPRHlb on the ASGPR-ASOR binding ability was tested by this cell line; the biological function of sASGPR was also studied by this cell line.Results1. The HBsAg targeting and shRNA expressing recombinant AAV2 virus was constructed successfully, designated rAAVssyU6-shRNA-hrGFP.The titer of the purified rAAVssyU6-shRNA-hrGFP was above 1×109v.g/ml.2. The efficiency of rAAVssyU6-shRNA-hrGFP infection on HepG2, HepG2.215 was increased, especially on HepG2.215. The HBsAg, HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAVssy-shHBs.3. The efficiency of the rAAVssyU6-shRNA-hrGFP on HepG2.215 cell was increased by heparin and decreased by HBsAb.4. The EGFP fusional protein vectors were constructed successfully. EGFP-Hla located on cell membrane; EGFP-Hlb expressed in cytoplasm by particle; EGFP-H2c expressed on cell membrane and in cytoplasm equably.5. A cell line which stably expression ASGPRHla and H2c was constructed successfully. The function of the cell line was confirmed by ASOR binding assay. Single Hlb showed no effect on ASOR binding to cells.sASGPR down regulated the ASGPR-ASOR binding.sASGPR could nonspecific bind to cells and ASOR specifically increased its binding. Conclusions1. The efficiency of rAAVssyU6-shRNA-hrGFP infection on HepG2.215 were increased markly.The HBsAg, HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAVssyU6-shRNA-hrGFP.2. rAAVssyU6-shRNA-hrGFP could target HBsAg but remain natural tropism.3. Single H1b showed no effect on ASGPR-ASOR binding.4. sASGPR could bind ASOR to form complex. SASGPR played a protective role in maintaining the balance of glycoconjugates metabolism.SASGPR recognized and bound the deleterious soluble glycoconjugates and then transported them safely to the liver, where they will be removed and degraded by the hepatic ASGPR system.Significances of the study1. This is the first reported rAAVssyU6-shRNA-hrGFP could targeting HBsAg and silencing HBsAg, HBeAg in vitro. This study laid a foundation for targeting gene therapy of HBV.2. Our works provide the first evidence of the function of human hepatic ASGPR Hlb and sASGPR in ASGPR-ligand binding. The study laid a foundation for further comprehensive study of the bionomics of ASGPR.