Progress With Developing Use of Gene Editing To Cure Chronic Infection With Hepatitis B Virus

Abstract

Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population. Carriers of the virus are at risk for life-threatening complications, and developing curative treatment remains a priority. The main shortcoming of licensed therapies is that they do not affect viral covalently closed circular DNA (cccDNA), a stable intermediate of replication. Harnessing gene editing to mutate cccDNA provides the means to inactivate HBV gene expression permanently. Reports have described use of engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) nucleases. Although inhibition of viral replication has been demonstrated, reliably detecting mutations in cccDNA has been difficult. Also, the dearth of murine models that mimic cccDNA formation has hampered analysis in vivo. To reach a stage of clinical use, efficient delivery of the editors to HBV-infected hepatocytes and limiting unintended off-target effects will be important. Investigating therapeutic efficacy in combination with other treatment strategies, such as immunotherapies, may be useful to augment antiviral effects. Advancing gene editing as a mode of treating HBV infection is now at an interesting stage and significant progress is likely to be made in the immediate future.

Figure 1

Schematic summary of the replication of HBV. (a) The small surface protein binds to glycosaminoglycans on the surface of hepatocytes. The virion attaches specifically and with high affinity to the sodium taurocholate cotransporting peptide receptor through interaction with the myristolated N-terminal region of the large surface protein. (b) After entry into the cytoplasm, probably by a process of endocytosis, the viral capsid containing relaxed circular DNA (rcDNA) is transported to the nucleus. (c) The rcDNA is released from the capsid and then (d) “repaired” to form covalently closed circular DNA (cccDNA). This stable and problematic replication intermediate is the target of strategies employing gene editing to disable HBV replication. (e) The cccDNA serves as template for transcription of viral pregenomic RNA (pgRNA) and protein-coding mRNAs. (f) Translation to form viral proteins leads to assembly of capsid particles containing packaged pgRNA, reverse transcription of this replication intermediate and (g) release via the secretory pathway. Licensed nucleoside and nucleotide analogs inhibit reverse transcription, and candidate therapeutic RNA interference activators disable viral RNAs. (h) Some rcDNA may be recycled from nascent core particles to generate cccDNA within the nucleus.

Figure 2

Arrangement of viral open reading frames and cis regulatory elements within the cccDNA of HBV. The cccDNA of HBV comprises approximately 3.2 kb. Nucleotide co-ordinates are typically calculated from the unique EcoRI site of the HBV DNA (top center). Approximate location of promoters, enhancers, and cis regulatory elements are indicated as circular and rectangular symbols on the cccDNA, which is shown as a double circle. The core (C), polymerase (P), surface (S), and X overlapping open reading frames are shown as arrows immediately surrounding the genome. Initiation of translation from the preS1 start codon generates the large surface antigen and the preS2 AUG begins synthesis of the middle HBsAg. The preC codon initiates formation of the secreted HBV e antigen (HBeAg).