One-Vector System for Multiplexed CRISPR/Cas9 against Hepatitis B Virus cccDNA Utilizing High-Capacity Adenoviral Vectors

Abstract

High-capacity adenoviral vectors (HCAdVs) devoid of all coding genes are powerful tools to deliver large DNA cargos into cells. Here HCAdVs were designed to deliver a multiplexed complete CRISPR/Cas9 nuclease system or a complete pair of transcription activator-like effector nucleases (TALENs) directed against the hepatitis B virus (HBV) genome. HBV, which remains a serious global health burden, forms covalently closed circular DNA (cccDNA) as a persistent DNA species in infected cells. This cccDNA promotes the chronic carrier status, and it represents a major hurdle in the treatment of chronic HBV infection. To date, only one study demonstrated viral delivery of a CRISPR/Cas9 system and a single guide RNA (gRNA) directed against HBV by adeno-associated viral (AAV) vectors. The advancement of this study is the co-delivery of multiple gRNA expression cassettes along with the Cas9 expression cassette in one HCAdV. Treatment of HBV infection models resulted in a significant reduction of HBV antigen production and the introduction of mutations into the HBV genome. In the transduction experiments, the HBV genome, including the HBV cccDNA, was degraded by the CRISPR/Cas9 system. In contrast, the combination of two parts of a TALEN pair in one vector could not be proven to yield an active system. In conclusion, we successfully delivered the CRISPR/Cas9 system containing three gRNAs using HCAdV, and we demonstrated its antiviral effect.

Host Transcription Factors in Hepatitis B Virus RNA Synthesis

“…CRISPR/Cas9, an accurate DNA editing technology can be used to mutate the cccDNA in order to destabilize the structure [285]. A recent study by Schiwon et al demonstrated that the CRISPR/Cas9-based system is capable to inhibit HBV replication [286]. However, the low concentrations of cccDNA in patients limit the effective targeting of CRISPR/Cas9 system, analysis of treatment, and potential off-targeting effects that affect the successful implementation of CRISPR/Cas9 system [285].…”

Hepatitis B virus persistence and reactivation

“…Among these, degrading cccDNA is an attractive strategy that is likely to cure occult HBV infection and abolish the risk of HBV reactivation. All candidates targeting cccDNA editing strategies, including zinc finger nucleases,184 transcription activator-like effector nucleases,185 and CRISPR/Cas9 systems186187 are effective in pre-clinical studies but have not yet been evaluated by clinical trials.…”

New Approaches to the Treatment of Chronic Hepatitis B

“…Hence, HBV cccDNA is an optimal target for nuclease gene editing, due to its episomal minichromosome configuration and sequence stability. More specifically, the efficacy of CRISPR/Cas9 for cleavage and inactivation of the cccDNA, as well as inhibition of hepatocarcinogenesis, has been reported [ 34 , 35 ].…”