Compositions and methods for treating viral infections
A virus, recombinant nucleic acid technology, applied in the field of compositions and methods for treating viral infections
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Embodiment 1
[0236] HepAD38 is a cell line that replicates human hepatitis B virus (HBV) under conditions regulated by tetracycline. In the presence of antibiotics, the cell line is virus-free due to inhibition of pregenomic (pg) RNA synthesis. Following removal of tetracycline from the medium, cells express viral pg RNA, accumulate subviral particles in the cytoplasm containing DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should facilitate analysis of the viral replication cycle that relies on the synthesis of viral DNA in a synchronized manner. Additionally, this cell line has been formatted as a high-throughput, cell-based assay that allows large-scale screening of various compound libraries for new classes of inhibitors of HBV replication. See Ladner S.K. et al. Antimicrob Agents Chemother. 1997 Aug;41(8):1715-20. Therefore, the HepAD38 cell line is a su...
Embodiment 2
[0241] Furthermore, in Figure 3A and Figure 3B as well as Figure 4A , Figure 4B and Figure 4C In experiment 2 shown in, the AAV8-HBV-DRS1 (EF1a>HBV-rcCasp9) construct was further tested in HepG2 cells and HepAD38 cells, and in some test groups, the caspase-9 inhibitor Z was used - LEHD-FMK to illustrate that killing is a result of Casp9 expression (see Groups 3 and 5). The results of these experiments illustrate virus-specific cell death in Group 2 HepAD38 cells, which exhibited almost 80% cell mortality. Figure 5A is the vector map, and Figure 5B is a combined graph showing the effect of the tested constructs and various controls in HepG2 cells and HepAD38 cells to highlight the severe cell death in HepAD38 cells infected with the AAV8-HBV-DRS1 (EF1a>HBV-rcCasp9) construct .
Embodiment 3
[0243] Finally, in experiment 3, the AAV8-HBV-DRS2 (TBG>HBV-rcCasp9) construct was tested. The construct contains a liver-specific promoter, thyroxine-binding globulin (TBG). The results of these experiments illustrate virus-specific cell death in Group 2 HepAD38 cells, which exhibited almost 80% cell mortality ( Figure 6A and Figure 6B ). In this grouping, cells in group 1 were not transduced, and cells in group 2 were test cells transduced with AAV8-HBV-DRS2 (TBG>HBV-rcCasp9); cells in group 3 were the same with the addition of the Casp9 inhibitor z-LEHD.fmk; group 3 Cells in group 4 were only exposed to the Casp9 inhibitor z-LEHD.fmk as a control; cells in group 5 were transduced with a GFP-containing construct to further follow construct expression; cells in group 6 were transduced with the same GFP construct and also treated with Casp9 The inhibitor z-LEHD.fmk was incubated together.
[0244] The test vehicle + Casp9 inhibitor control group in experiments 2-3 indica...
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