Gene Therapy for Chronic HBV—Can We Eliminate cccDNA?

Bloom, Kristie; Maepa, Mohube Betty; Ely, Abdullah; Arbuthnot, Patrick

Natural epigenetic modification of DNA may silence gene expression and is a host defence mechanism against expression of viral genes. Epigenetic modification is a stable and heritable gene regulatory mechanism found in many different organisms. It involves chemical alteration of DNA or associated proteins without changing the encoded genetic information. It is involved in typical cell development and multiple normal or abnormal cellular responses (reviewed by [69] ). There is accumulating evidence to indicate that epigenetic machinery controls transcription of HBV cccDNA ( Figure 1B) , and contributes to the outcome of chronic HBV infection [70] [71] [72] . Use of exogenous epigenetic modifiers has thus attracted interest as a mode of disabling cccDNA. Epigenetic modifications include histone acetylation or deacetylation, histone methylation or demethylation, cccDNA methylation, and cccDNA minichromosome acetylation (reviewed by [73, 74] ). In cooperation with viral factors such as HBx and the core antigen (HBc), the major epigenetic modifiers of HBV DNA include histone acetyltransferases/deacetylases (HATs/HDACs) [12] , lysine methyltransferases [75] , protein arginine methyltransferases [70, 76] , and DNA methyltransferases (DNMTs) [77] . Hypoacetylation of cccDNA-bound histone 3/4 results in low HBV viremia in hepatitis B patients, whereas hyperacetylation increases HBV replication [11] . HDAC inhibitors have been shown to suppress cccDNA transcription in duck hepatitis B virus (DHBV) [82, 83] . Methylation of arginine 3 on cccDNA-bound histone 4 prevents RNA polymerase binding and transcription in an arginine methyltransferase 5 and HBc-dependent manner [70, 76] . Transcriptional inhibition by methylation can be direct, by blocking binding of transcriptional factors or RNA-polymerase loading, as well as indirect, by recruiting histone-modifying and chromatin remodelling complexes to the methylated DNA (reviewed by [69] ). Epigenetic gene silencing may facilitate reduction of viral reservoirs through normal hepatocyte turnover and prevention of replenishment of the cccDNA pool. Furthermore, epigenetic modifications may accelerate cccDNA decay, as was shown following IFN-α treatment of DHBV infections [82] . HBV cccDNA contains three CpG islands: island I, island II and island III [84, 85] . Island I overlaps the start site of the S gene, island II encompasses enhancer I, the HBx gene promoter and the core promoter, whereas island III harbors the Sp1 promoter and start codon of the polymerase gene. Methylation of island I is rare and variable across genotypes, whereas methylation of island II and III is more conserved. Island III methylation is associated with reduced serum HBsAg concentrations in chronically infected patients and correlates with hepatocarcinogenesis. Island II methylation is associated with reduced pregenomic RNA (pgRNA) transcription, viral replication and viremia [72, 86] . In vitro studies showed that several DNMTs are upregulated in response to HBV infection, leading to viral DNA methylation, decreased HBV gene expression, and diminished viral replication [77, 87] . Importantly, similar reduction in viral gene expression and viremia was observed in human tissue samples with methylated HBV DNA [85, 88, 89] .