Development of hepatitis B virus as vectors for gene therapy

Hepatitis B virus (HBV) is attractive for application as gene delivery vectors for gene therapy of liver diseases because of its liver specificity. In this study, the feasibility of using HBV as replicative and nonreplicative viral vectors for liver gene transfer has been investigated.; An HBV replicative vector was developed by insertion of a foreign gene, the tat gene from the human immunodeficiency virus type 1 (HIV-1), into the tether region in-frame with polymerase gene (P) of the HBV genome. Expression of a functional tat protein (Tat) by this HBV construct was demonstrated by its transactivation activity on the HIV-1 long terminal repeat. Expression of Tat in chicken hepatoma and human cervical carcinoma cells was significantly lower than that expressed in human hepatoblastoma. cells, thus indicating the cell type and species specificity of HBV. Expression of the functional Tat by this HBV replicative vector seemed to be regulated by the preS1 promoter and/or the C promoter. Replication competence of this HBV replicative vector was retained but at a reduced level of about 1.5% of the HBV wild type capability. Complete viral particles was produced from this vector.; Application of this HBV replicative vector for gene transfer appears to be limited by the size and the functional conformation of a foreign gene to be expressed. The ZeocinTM resistant gene (ZeoR), a reporter gene about 100 base pairs larger than the tat gene, could not be expressed by an HBV replicative vector, possibly due to the inability of the ZeoR protein to function in a fusion form.; An HBV nonreplicative vector was developed by insertion of the Zeo R gene with a stop codon into the HBV genome using the same strategy used for construction of the HBV replicative vector. Functional Zeo R protein was expressed by this vector. Replication of this HBV nonreplicative vector could be supported by trans-complementation with the polymerase protein.; The chicken anemia virus VP3 gene (CAV-VP3) or apoptin, which has potential use in cancer gene therapy, was expressed by both HBV replicative and nonreplicative vectors as detected by its apoptotic inducibility in human hepatoblastoma cells. However, the activity of the CAV-VP3 protein expressed from the HBV replicative vector seemed to be lower than that expressed from the HBV nonreplicative construct.; The infectivity of HBV recombinant particles produced from HBV vectors was also investigated using the in vitro infection assay presented by Lu and colleagues (J Virol 1996; 70: 2277--2285). Study on the infectivity of HBVtat recombinant particles indicated that the HBVtat vector particles are infectious.