Targeting HBV cccDNA Levels: Key to Achieving Complete Cure of Chronic Hepatitis B

Abstract

Chronic hepatitis B (CHB) caused by HBV infection has brought suffering to numerous people. Due to the stable existence of HBV cccDNA, the original template for HBV replication, chronic hepatitis B (CHB) is difficult to cure completely. Despite current antiviral strategies being able to effectively limit the progression of CHB, complete CHB cure requires directly targeting HBV cccDNA. In this review, we discuss strategies that may achieve a complete cure of CHB, including inhibition of cccDNA de novo synthesis, targeting cccDNA degradation through host factors and small molecules, CRISP-Cas9-based cccDNA editing, and silencing cccDNA epigenetically.

Keywords: CRISP-cas9; HBV; cccDNA; chronic hepatitis B; interferon.

Figure 1

HBV lifecycle. HBV first binds to the low-affinity receptor HSPG, then interacts with NTCP, and enters the liver through endocytosis with the support of EGFR. After removing the nucleocapsid, rcDNA enters the nucleus through the nuclear pore and is subsequently stripped of the polymerase that binds to it, forming DP-rCDNA with the assistance of TDP2. Then, DP-rcDNA utilizes host factors to repair double-stranded gaps and form cccDNA. cccDNA forms minichromosomes with histones and other proteins and is transcribed into HBV transcripts, including pgRNA, with the help of HBx and epigenetic factors. These transcripts are then translated into several viral proteins. pgRNA forms rcDNA through reverse transcription and assembles into virions with viral proteins. Virions are then expelled through exocytosis.

Figure 2

Approaches targeting HBV cccDNA levels. CKII inhibitor DMAT prevents the nuclear import of the polymerase–rcDNA complex. Disubstituted sulfonamides (DSS), CCC-0975 and CCC-0346, disrupt the production of DP-rcDNA and then inhibit cccDNA formation. ATR inhibitors, DNA ligases inhibitors, DNA polymerase inhibitor aphidicolin, FEN1 inhibitor PTPD, and PARP inhibitor Olaparib block the conversion of rcDNA to cccDNA. APOBEC3A/B, UNG2, ISG20, and APEX1 may collaborate to degrade cccDNA. cccDNA reducers, like ccc_R08, decrease cccDNA levels, though their precise mechanisms remain unknown. Although CRISPR-Cas9 KO targeting cccDNA enhances the clearance of cccDNA, it has the potential for off-target effects and poor delivery efficiency. A CRISPR/Cas9-mediated non-cleaving base editor that introduces nonsense mutations into cccDNA is a more promising approach. Epigenetic silencing of cccDNA is another approach, but the numerous epigenetic factors regulating cccDNA minichromosomes present a challenge. A more effective target may be the HBx protein, siRNA, or small-molecule compounds targeting HBx could effectively inhibit cccDNA transcription. ➀ CKII inhibitor DMAT. ➁ Disubstituted sulfonamides (DSS). ➂ ATR inhibitors, DNA ligases inhibitors, DNA polymerase inhibitor, FEN1 inhibitor, PARP inhibitor. ➃ APOBEC3A/B, UNG2, ISG20, APEX1, cccDNA reducers. ➄ CRISPR-Cas9 KO targeting cccDNA, CRISPR/Cas9-mediated non-cleaving base editor. ➅ SETDB1, PRMT5, PRMT1, SIRT3, HMGA1, SMC5/6. ➆ siRNA targeting HBx, Dicoumarol, Nitazoxanide, Estradiol Benzoate, MLN4924 (pevonedistat). The red cross indicates inhibition and the red arrow indicates promotion.