Role of hepatitis B virus in development of hepatocellular carcinoma: Focus on covalently closed circular DNA

Table 1 Methods to detect covalently closed circular DNA

Method	Advantages	Disadvantages	Ref.
Blotting
Southern blot	Specific detection of DNA sequences; Able to distinguish cccDNA from other viral DNA species; Reliable and reproducible	Complicated, time-consuming, and costly	[101-102]
PCR-based methods
Conventional qPCR	Simple, accurate, and sensitive; Suitable for high-throughput screening; Rapid and economical	Specificity is reduced if high concentration of rcDNA is present	[100,102]
Semi-nested and nested qPCR	Sensitive and specific; Allows for cccDNA quantification		[100,102,104,106]
Competitive qPCR	Sensitive; Can readily distinguish cccDNA from rcDNA; Allows for cccDNA quantification		[100,102,107]
Droplet-digital PCR	Very sensitive and accurate; Can detect a single copy of cccDNA precisely; Allows for cccDNA quantification		[102,108-114]
Rolling circle amplification qPCR	Very sensitive; cccDNA is visible at single-cell resolution	Effective amplification may be hindered by cross-linked proteins or diffusion of DNA	[100,102,115-117]
Magnetic capture hybridization PCR	Sensitive and specific; Allows for selective isolation of cccDNA; Reproducible	Unable to capture all cccDNA completely; Complicated and costly	[100,102,118-119]
Invader Assay	Specific, simple, and reproducible		[100,102,121-124]
In situ hybridization	Specific; Can distinguish different types of nucleic acids; Visible at single-cell resolution	Complicated probe design	[100,102,125-126]
Indirect method
Surrogate markers	Non-invasive; Convenient and cost-effective; Suitable for high-throughput screening	Indirect	[100,114,126-128,131-135]

cccDNA: Covalently closed circular DNA.

Table 2 Gene editing and epigenetic modification techniques to target and eliminate covalently closed circular DNA

Technique	Study model	Study results	Ref.
Gene editing
Synthetic RNAi	Clinical trial	ARC-520 was well tolerated, with only two serious adverse effects. ARC-520 was active in both HBeAg-neg and HBeAg-pos patients, but only moderate reduction in HBsAg was observed	[122]
Zinc finger nucleases (ZFNs)	In vitro (AAV-mediated delivery of ZFNs in HepAD38 cells)	Completely inhibited HBV DNA replication and decreased HBV pgRNA level	[124]
	In vitro	Decreased pgRNA level, thus having the potential to target cccDNA	[123]
Transcription activator-like effector nucleases (TALENs)	In vitro and in vivo [murine hydrodynamic injection (HDI)]	Efficient disruption of target sites and suppression of viral replication markers; targeted mutation in 35% of cccDNAs was observed in vitro under mildly hyphothermic conditions and further confirmed in vivo	[125]
CRISPR/Cas9 System	In vitro (A64 cells)	Inhibited both HBV antigen expression and replication, excised the entire full-length of integrated HBV genome, and disrupted cccDNA	[126-131]
Epigenetic modification
Dicoumarol	In vitro (HBV-infected cells HepG2-NTCP cells) and in vivo (humanised liver mouse)	Reduced HBx protein expression, therefore having a potent antiviral activity against HBV RNAs, DNA, HBsAg, and HBc protein; cccDNA-ChIP decreased active histone marks and increased repressive histone marks	[132]
	In vitro (NTCP-expressing HepG2 and primary hepatocytes)	Inhibited HBV replication in HBV-infected primary human hepatocytes by inhibiting the activity of cccDNA	[133]
Interferon-alpha (IFN)	In vitro and in vivo (chimeric uPA/SCID mice)	Hypoacetylation of cccDNA-bound histone and active recruitment of transcriptional corepressors to the cccDNA; Inhibited HBV replication and cccDNA transcription	[134]
	In vitro	Induced a prolonged suppression of human and duck HBV cccDNA transcription	[135]
Zinc finger proteins (ZFPs)	In vitro (male longhorn hepatoma cells)	ZFPs binding to HBV enhancer region inhibited viral replication by inhibiting cccDNA transcriptional activity	[136]
Curcumin	In vitro (HepG2.2.15)	Reduced HBsAg and cccDNA levels up to 58% and 76%, respectively	[137]

cccDNA: Covalently closed circular DNA; HBV: Hepatitis B Virus; HBx: HBV X; ZFPs: Zinc finger proteins.