Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR/SaCas9 Expressed From Liver-Specific Promoters

Abstract

Curative therapies for chronic hepatitis B virus (HBV) infection remain a distant goal, and the persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is a key barrier that is hard to break through using the drugs currently approved for HBV treatment. Due to the accuracy, efficiency, and cost-effectiveness of genome editing, CRISPR/Cas technologies are being widely used for gene therapy and in antiviral strategies. Although CRISPR/Cas could possibly clear cccDNA, ensuring its safety is requirement for application. In our study, we analyzed the liver specificity of several promoters and constructed candidate promoters in the CRISPR/Staphylococcus aureus Cas9 (SaCas9) system combined with hepatotropic AAV8 (whereby AAV refers to adeno-associated virus) to verify the efficacy against HBV. The results revealed that the reconstructed CRISPR/SaCas9 system in which the original promoter replaced with a liver-specific promoter could still inhibit HBV replication both in vitro and in vivo. Three functional guide RNAs (gRNAs), T2, T3, and T6, which target the conserved regions of different HBV genotypes, demonstrated consistently better anti-HBV effects with different liver-specific promoters. Moreover, the three gRNAs inhibited the replication of HBV genotypes A, B, and C to varying degrees. Under the action of the EnhII-Pa1AT promoter and AAV8, the expression of SaCas9 was further decreased in other organs or tissues in comparison to liver. These results are helpful for clinical applications in liver by ensuring the effects of the CRISPR/Cas9 system remain restricted to liver and, thereby, reducing the probability of undesired and harmful effects through nonspecific targeting in other organs.

Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

“…CRISPR/Cas also has the advantage that multiple sites on the viral genome can be targeted by increasing the number of gRNAs, rather than needing to express two or more enzymes. CRISPR-based genome editing been used to target viruses including hepatitis B virus (HBV) [85,86], human immunodeficiency virus (HIV) [87,88], and several herpesviruses [89] such as EBV [90], CMV [91], Kaposi's Sarcoma Virus [92], and HSV-1 [31][32][33]. In the case of EBV, antiviral gene editing has targeted both the viral genome [90] as well as key cellular components required for EBV replication [93].…”

The Application of CRISPR/Cas Systems for Antiviral Therapy

“…Type BmNPV: (Chen et al, 2017), (Dong et al, 2018a), (Dong et al, 2018b), (Dong et al, 2019a), (Dong et al, 2019b) Hepatitis B virus: , (Kostyusheva et al, 2019), (Li et al, 2018), (Liu et al, 2018), (Schiwon et al, 2018), (Stone et al, 2021), (Suzuki et al, 2021), (Yan et al, 2021), (Yang et al, 2020) HIV-1: (Bella et al, 2018), (Dash et al, 2019), (Ebina et al, 2013), (Hu et al, 2014), (Kaminski et al, 2016a), (Kaminski et al, 2016b) Finally, we note that RNA interference (RNAi) for silencing viral gene expression is a possible alternative therapy. RNAi, like Cas13, also offers a sequence-targeted antiviral approach in this case by binding to and degrading specific RNA transcripts (Levanova and Poranen, 2018).…”

“…Moreover, to address safety concerns, the activity of CRISPR/Cas9 against HBV can be limited to the liver by using AAV8 which shows high liver tropism. Liver-specific promoters controlling the expression of CRISPR/Cas9 from AAV8 are effective as an additional method to restrict activity to the liver, as demonstrated in mice ( Yan et al, 2021 ).…”

The State-of-the-Art of Gene Editing and its Application to Viral Infections and Diseases Including COVID-19

“…Therefore, gene therapy that targets cccDNA holds a potential therapeutic avenue to attain complete clinical curing of CHB infection ( Maepa et al., 2015 ; Bloom et al., 2018 ). Multiple studies have investigated the application of CRISPR/Cas9 for targeting and cleavage of many essential loci of HBV such as cccDNA, surface antigen, and X gene in cell cultures or animal models ( Seeger and Sohn, 2014a ; Kennedy et al., 2015 ; Ramanan et al., 2015a ; Zhen et al., 2015 ; Yan et al., 2021 ; Martinez et al., 2022 ). Seeger and Shon demonstrated the first use of the CRISPR/Cas9 strategy to disrupt the HBV cccDNA.…”