[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
4.1 Construction of retroviral vectors and producer lines encoding an antisense and sense copy of surface antigen
Recombinant clones encoding HBsAg gene were identified by colony hybridization with a 32P-labeled probe. Plasmid DNA was isolated from each positive clone and subjected to restriction enzyme analysis. The clones which released the 0.8 Kb fragment after XhoI digestion were further digested with SspI. The clones (named pYT7+) containing the HBsAg gene in the sense orientation released 6.2 and 3.3 Kb bands. The clones containing antisense HBsAg (named pYT7-) released 5.5 and 4.1 Kb bands.
4.2 Inhibition of HBsAg production by co-transfection assay
HepG2 cells were co-tranfected with pYT7- and pTHBV at molar ratios of 1:1 and 1:2 by calcium phosphate method. When a higher molar ratio was used, lower transfection efficiency was observed. Because HBsAg was detectable in pYT7+ transfected HepG2 cells, only vector (pMNC) was used as control DNA for co-transfection. The inhibition of HBsAg expression was measured by ELISA as described in Materials and Methods.
The results indicated that the expression of HBsAg was inhibited by 45-55 % in antisense construct (pYT7-) co-transfected cells as compared to vector (pMNC) co-transfected cells (figure 2). The data shown represents the average of three independent (duplicate) experiments.
Figure 2. Inhibition of HBsAg expression pYT7- was co-transfected with pHBV in the molar ratio of 2 and 1. The level of HBsAg was compared to the cells co-transfected with pTHBV and pMNC.
4.3 Inhibition of HBsAg production in the antisense-transduced HepG2 cells
It is possible that multiple copies of antisense constructs and HBV plasmids could enter single co-transfected cells in previous transient co-transfection assays. To test whether the inhibition of surface antigen expression will still take place in hepatocytes transduced with single copies of antisense gene, the human hepatoma cell line HepG2 was transduced with cell-free virus harvested from the producer lines of MNC and YT7- having the highest titers. Transduced cells were selected in the presence of G418 (750 mg/ml) for 14-16 days. The resulting G418 resistant colonies were pooled and expanded to a cell line. The presence of HBsAg gene in the transduced cells was comfirmed by PCR (data not shown). The transduced HepG2 cells were analyzed for antisense RNA expression by Northern analysis. The results indicated that two antisense RNA transcripts (3.8 and 1.8 kilobases) were driven by LTR and CMV promoters, respectively (figure 3). Both RNA transcripts, including approximately 1 kilobase RNA, were derived from the vector and poly A signal. The YT7- and MNC transduced cells were challenged with HBV by transfecting 5 mg of pTHBV and subsequently measuring the levels of ELISA-detectable HBsAg products secreted into the growth medium as previously described. The results showed that HepG2 cells transduced with the antisense HBsAg construct resulted in an approximate 75% inhibition in HBsAg expression as compared to the control cells, MNC transduced cells 3 days after transacting with pTHBV (figure 4).
Figure 3. Northern analysis of transduced HepG2 cells. Total cellular RNA was extracted from transduced cells and probed with 32p labeled HBsAg or Neo gene. Lane 1: retroviral vector MNC, lane2: sense copy of HBsAg, lane3: antisense copy of HBsAg retroviral transduced cells. <
Figure 4. Inhibition of HBsAg expression in the transduced HepG2 cells. Transduced HepG2 cells were transfected with 5 mg of pTHBV. The data shown is an average of 6 assays.