CccDna Directed Therapy Host Factors Implicated in cccDna Biogenesis

To date, the process of cccDNA biogenesis is not fully understood, but it is conceivable that several host factors are exploited by HBV for cccDNA biogenesis. These could serve as potential candidates for cccDNA-targeted therapies.

TDP2

RC-DNA must be subjected to multiple manipulations in order to transform into cccDNA, rendering it very likely to interact with the host DNA repair machinery. Tyrosyl-DNA-phosphodiesterase 2 (TDP2) is a DNA repair enzyme that has been shown to specifically cleave a tyrosine-DNA bond in duck HBV (DHBV) RC-DNA releasing the viral polymerase (P protein), a critical step in converting RC-DNA into cccDNA. Knocking-down TDP2 in human cells significantly decelerated the conversion of RC-DNA to cccDNA, while ectopic TDP2 expression in the same cells restored faster conversion kinetics.^[70] This study identifies for the first time a cellular factor implicated in cccDNA biogenesis. Of note, in a recent study Cui et al. demonstrated that although TDP-2 cleaves the tyrosyl-minus strand DNA linkage in vitro,TDP2 gene knockout in human hepatoma cells did not block cccDNA formation.^[71]These findings suggest that TDP2 is not absolutely required for cccDNA formation and calls into question the utility of TDP2 as a target for cccDNA elimination.

Targeting hbv dna With the crispr/Cas9 System

CRISPR (clustered regularly interspaced short palindromic repeats) loci, combined with CRISPR associated protein (Cas) genes, are found in the genome of numerous bacteria. The CRISPR machinery has been found to recognize, attach to and cleave different DNA sequences of invading bacteriophages through complementary sequencing, rendering the offended DNA for degradation.^[72] As the episomal DNA of HBV is a major target for therapy, recent studies have attempted to design CRISPR constructs that target conserved regions along the viral genome. Those studies have shown that CRIPSR/Cas9 not only suppresses viral gene expression and replication but also eliminates a substantial fraction of intracellular cccDNA.^[73–77] Developing efficient ways for delivery of the CRISPR/Cas9 constructs to the liver remains the Achilles heel in further implementation of this method. A recent report utilizing Cas9 from Staphylococcus aureus (SaCas9) for efficient adeno-associated virus (AAV) vehicle-based delivery to the liver^[78] is an encouraging step forward in this direction.

Small Molecules Targeting cccDna

Based on a specialized cell line dependent solely on cccDNA for HBV e antigen (HBeAg) production (HepDE19 cell line), Cai et al. screened a library of 85 000 small molecules to reveal two disubstituted sulfonamides (DSS) compounds able to inhibit cccDNA formation and/or maintenance.^[79]Interestingly, in contrast to NUCs, these DSS compounds blocked the conversion of HBV rcDNA into cccDNA rather than inhibiting the production of rcDNA, resulting in reduced levels of both viral cccDNA and deproteiniezed rcDNA (DP-rcDNA), the cccDNA precursor.^[80] Furthermore, DSS exerted their effect on the cccDNA pool without directly influencing viral DNA replication in both HBV and DHBV models. However, one must take into account that mechanisms implicated in DHBV cccDNA formation and amplification may not apply to HBV, as suggested by studies done in the HepaRG cell line.^[81] In addition, the HepDE19 cell line system might not be specific enough for cccDNA detection and therefore results should be interpreted with caution until better reporters for cccDNA are developed.