Introduction:
There is a limit to the effect of current therapies for chronic hepatitis B virus (HBV) infection on viral gene expression and replication. However, improved technology has allowed induction of RNA interference (RNAi) in mammalian cells by using short interfering RNA duplexes (siRNA). This is a new approach to inhibit HBV infection and replication.
During RNAi, sequence-specific silencing of homologous genes is directed by double-stranded RNA. The HepG2.2.15 cell line, which is a derivative of the human HepG2 hepatoma cell line which has been stably transformed with several copies of the HBV genome, has been used to study the effect of RNAi on HBV replication. This line serves as an in vitro model for HBV replication.
Method:
1. Purify the endotoxin-free plasmid DNA using the EndoFree Plasmid Kit
2. Grow HepG2 and HepG2.2.15 cells in Dulbecco's modified Eagle's medium (DMEM)
3. Transfect cells in the presence of siRNA at 70% confluency
4. Inject plasmid DNA and siRNAs into mice via the tail vein
5. Disect the liver of the mice into pieces and freeze them immediately for preservation
6. Stain the tissue section of the formalin-fixed mice livers with hematoxylin and eosin
Northern Blot Analysis:
7. Digest total RNA extracted from the cells with DNase
8. Separate RNA with agarose gel electrophoresis
9. Probe the blots with 32P-labeled HBV DNA
Southern Blot Analysis:
7. Load samples containing the entire amount of isolated DNA onto an agarose gel
8. Transfer the gel to a GeneScreenPlus membrane
9. Hybridize the gel with a 32P-labeled HBV cDNA probe
10. Extract DNA from serum of the mice and subject them to Polymerase Chain Reaction (PCR)
11. Dot blot the PCR mixture
12. Hybridize the mixture with a 32P-labeled 424bp DNA fragment
13. Quantitate the viral particles in mice sera by comparing the spot intensities with a standard curve
Results and discussion:
It is proven that HBV gene expression and viral replication in both HepG2.2.15 cells and in vivo in a mouse model can be inhibited by the double-stranded RNA duplex siRNA-1 directed against HBV sequences in the s antigen/polymerase region. Besides inhibiting viral RNA, siRNA-1 had a dramatic effect on viral DNA production, rendering HBV DNA undetectable in sera of treated mice within 2 days after injection.
The effect of siRNA-1 on viral DNA production is indirectly caused by the suppression of both the RNA template and the polymerase enzyme, which are essential for viral DNA synthesis.
The advantage of using RNAi in the therapy of HBV is that the effect appears to be specific for the targeted gene and side effects are minimized. siRNAs simultaneously targeted to different regions of the virus may be a way to increase the efficiency of the treatment and prevent the appearance of viral revertants resistant to the treatment.
Conclusion:
In conclusion, siRNA can induce an antiviral effect on HBV replication and gene expression both in vitro and in vivo. This is useful in future studies that will assess the therapeutic potential of siRNAs for treatment of acute and chronic infections by a pathogen.
Scientific journal:
http://www.nature.com/mt/journal/v8/n5/full/mt2003238a.html
