[Frontiers in Bioscience 3, a11-15, February 1, 1998]|
TARGETED INHIBITION OF HEPATITIS B VIRUS GENE EXPRESSION: A GENE THERAPY APPROACH
Frank Y.-T. Tung and Steven W. Bowen
Department of Infectious Disease and Microbiology, University of Pittsburgh, Pittsburgh PA 15261
Received 1/21/98 Accepted 1/28/98
3. MATERIALS AND METHODS
3.1. Construction of retroviral vectors and producer lines
We chose HBsAg gene as target for antisense RNA mediated inhibition because the HBsAg has been identified as target antigen for cytotoxic T lymphocytes (27,28) and the quantitative ELISA kit for HBsAg expression is commercially available. The Moloney murine leukemia virus-based expression vector pMNC (figure 1) was used to construct antisense and sense constructs. A PCR product comprising the HBsAg gene (820 bp) was prepared using a plasmid containing single copies of the complete genome of HBV (pTWL1) as the template DNA (29). Synthetic oligonucleotides (FT7:5' GGATCCTCGA GTCAGGGGTCTGTA and FT8:5'GTTTCTCGAGGGTT T AAATGTATACCCA) corresponding to 5' and 3' ends of HBsAg gene were used for PCR. An additional XhoI restriction site was added to primers to facilitate subcloning. The amplified fragment was digested with XhoI, then ligated into XhoI-linearized murine retroviral vector pMNC (30). The recombinant clones containing the HBsAg sequence were identified with 32P-labeled HBsAg probe. The orientation of the insert was confirmed by restriction enzyme analysis. pYT7- contains a copy of HBsAg in the antisense orientation and pYT7+ contains a copy of HBsAg in the sense orientation. Plasmid DNA was purified by CsCl ultracentrifugation. Virus producer lines were generated as described (31). In brief, 5 mg DNA was transfected into packaging cell line GP+envAm12 by electroporation. Individual stable transfected cells were selected in 500 mg/ml G418 (75% active, Life Technologies, USA). The viral titers of cell-free viruses from 30 clonal cell lines were determined on NIH3T3 cells as described (31). A high-titer producer line (5x106 colony forming unit/ml) was selected for further transduction experiments.
Figure. 1. Construction of recombinant retroviral vector. Two RNA transcripts (3.8 and 1.8 kilobases) encoding HBsAg were driven by Long Terminal Repeat (LTR) and cytomegalovirus (CMV) early promoters, respectively (also see Fig.3). The Neo resistant gene was driven by the LTR promoter.
3.2. Transient assay of antisense-mediated inhibition of HBsAg
The hepatoma cell line, HepG2, was co-transfected with HBV molecular clone (pTHBV,32) and pYT7- or vector control (pMNC) at different molar ratios by calcium phosphate precipitation method. Co-transfected cells were incubated for approximately 10.5 hrs, after which they were rinsed with PBS and the cells further incubated in growth medium for 68 hrs. Samples were then collected for assay of HBsAg secreted into the medium. The medium taken from the dish was centrifuged briefly to remove any dead cells and debris, and stored at -20 °C until ready to be used. At the time of assay, 1 ml samples were subjected to evaporation by Speed Vac to 0.1 ml (10-fold concentration) and added into anti-HBsAg antibody-coated wells using a commercially available ELISA kit (United Biochemical Inc., Mountain View, CA, USA). The antibody-antigen-antibody conjugate sandwich assay system was performed according to the manufacturer's protocol.
3.3. Northern blot analysis of antisense and sense RNA expressed in transduced HepG2 cells
Total RNA was extracted from transduced HepG2 cells with an RNA Extraction Kit (Stratagene, La Jolla, CA, USA). RNA was fractionated on a 1.2 % formaldehyde gel and capillary blotted onto charged nylon paper (MicroSeparation Inc. Westboro, MA, USA). The blot was probed with a 32P-labeled HBsAg DNA fragment prepared by PCR.