Introduction:
Globally, chronic hepatitis B virus (HBV) infection, contributes to 1 million hepatocellular carcinoma and liver disease related deaths annually. Chronicity of infection is largely due to a reservoir of HBV covalently closed circular DNA (cccDNA), which acts as the central transcription and replication template. cccDNA encodes the multifunctional HBV X-protein (HBx) which maintains cccDNA stability. HBx also interacts with host proteins, including DDB1, to degrade the host Structural Maintenance of Chromosomes 5/6 (Smc5/6) complex, which otherwise directs cccDNA towards epigenetic silencing.
We hypothesise that by knocking down HBx expression using CRISPR-Cas9 base editor and CRISPR-Cas13b systems, targeting DNA and RNA respectively, we will observe decreased levels of cccDNA and an increase in Smc5/6 protein. To quantify cccDNA, a digital PCR assay will be developed and the level of HBx loss required for Smc5/6 recovery will be investigated.
Methods:
For dPCR assay development, different primer-probe combinations, concentrations and annealing temperatures were assessed. To measure the effect of HBx on cellular Smc5/6 expression, HepG2 cells were transfected with wildtype HBx over-expression plasmid (pCIX) and a number of mutant HBx plasmids. Smc5/6 expression was determined in the cell lysates by western blotting.
Results:
Serial dilutions of a HBV expressing plasmid were used to establish sensitivity and specificity of the digital PCR. Differences in Smc6 levels in samples transfected with wildtype HBx and mutant HBx were observed, with the presence of HBx resulting in decreased Smc6 levels.
Discussion:
The digital PCR assay now allows quantification of cccDNA in extracted cell culture and liver samples. Transfection studies using overexpression systems showed that HBx impacts Smc5/6 levels. For future experiments, the impacts of HBx knockdown on cellular Smc5/6 levels, HBV cccDNA expression and HBV transcription will be investigated using an array of models.