A Trial Study On Transforming Hepatitis B Virus As The Liver-targeting Gene Therapeutic Vector

ObjectivesHepatitis B virus (HBV) is naturally provided with the prerequisite for being transformed as the liver-targeting gene therapeutic vector because of its hepatotropism specificity. It can specifically infect the liver and continuously replicate in the hepatocytes without obvious cytotoxicity by itself. Moreover, it has also the capability of carrying, transferring and expressing the gene of interest, which is ultimately encapsidated into the viral particles. However, it is a pity that there are many shortcomings in the present developed HBV-based vectors such as the poor capability of carrying long foreign gene, the low efficiency of replication and encapsidation, the potential risk of immunogenicity etc, which all result from the complex genomic structure of HBV. In view of the biological features of the naturally occurring defective HBV mutants obtained from the hepatitis patients, the C gene-truncated HBV vectors were experimentally constructed in this study as well as investigated for their molecular biological features including the expression, replication and encapsidation etc. in order to seek for the ideal strategy for developing the HBV-based vector. MethodsThe recombinant HBV vector of pHBV-S-GFP carrying the gene of interest was constructed in which a fragment encompassing the S gene was replaced by a DNA fragment encoding GFP fused to the first three amino acids of S. After the recombinant plasmid of pHBV-S-GFP was solely transfected to HepG2 by using the liposome method, the expression of GFP was observed with the fluorescence microscope. At the same time the plasmid of pCMV-GFP was selected as the positive control in parallel, which could effectively express the green fluorescence protein in the liver cells. After the plasmid of pHBV-S-GFP was co-transfected with the helper construct of pHBV3142 devoid of the encapsidation signal ε, the viral DNA were1. extracted from the transfected HepG2 nucleolus about 48 hours later. The rHBV cccDNA was testified by using Semi-Nested-PCR with the specific primers designed to encompass both GFP and HBV DNA sequence in terms of the characteristic of HBV replication. The extract from culture medium was detected by PCR for both GFP and HBV DNA in order to testify the possibility that the "therapeutic" rHBV progeny particles carrying the GFP gene could be encapsidated to secrete into the supernatant. The encapsidation efficiency of the rHBV vector was evaluated by southern blot assay in which the wild plasmid pHBV3091 was used as control.2. The C-truncated HBV mutants, pHBV-C△188( 220-407nt) and pHBV-C△141(267-407nt), were constructed by the molecular cloning and PCR-based deletion from the wild HBV plasmid of pHBV3091.The DNA of virus mutants were extracted from the cytoplasm and supernatant after the mutants plasmids were transfected solely or co-transfected with the helper construct of pHBV3142 devoid of the encapsidation signal ε.The replicating and encapsidating capabilities of both C-truncated HBV mutants were investigated by southern blot assay. These replication intermediate ccc DNA of both mutants extracted from the nucleolus of transfected HepG2 were testified by using Semi-Nested-PCR with the specific primers which were designed in terms of the characteristic of HBV replication. These mutants DNA extracted from the extracelluar supernatant were analyzed by using the Real-time fluorescence quantitative PCR in order to compare the encapsidation efficiency between the C-truncated HBV mutants and the wild HBV. The mutants RNA were extracted from the transfected HepG2 cells to evaluate quantitatively the effect on HBV RNA transcription in both C-truncated HBV mutants by using the Real-time fluorescence quantitative RT-PCR. The S protein expression was detected from the cytoplasmic lysates by western blot assay and further analyzed quantitatively by ELISA to determine the effect of the truncated C gene on S protein expression.The C-truncated rHBV vectors carrying the reporter gene of GFP were constructed as pHBV-C△188-S-GFP and...