Hepatitis B virus (HBV) is a major global health problem, affecting about 257 million people worldwide and causing chronic liver disease, cirrhosis, and hepatocellular carcinoma. Despite the availability of effective vaccines and antiviral drugs, HBV infection remains incurable, mainly due to the persistence of a viral episomal DNA, known as covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes. The cccDNA serves as the template for the transcription of all viral RNAs and is responsible for the high rate of viral rebound after cessation of therapy. Therefore, targeting the cccDNA is considered a key strategy to achieve a functional cure for HBV infection.
Fig.1 Hepatitis B virus life cycle along with inhibitors targeting the various stages of
the hepatitis B virus lifecycle. 1
Gene and epigenome editing technologies have emerged as promising tools to manipulate the HBV genome and epigenome and thus inhibit its replication and transcription. Gene editing technologies, such as nucleases and base editors, can introduce targeted cuts or mutations in the HBV cccDNA and integrated DNA, resulting in their degradation or inactivation. Epigenome editing technologies, such as epigenetic modifiers, can alter the epigenetic status and transcriptional activity of the HBV cccDNA, which is influenced by DNA methylation and histone modifications. These technologies have shown promising results in vitro and in vivo, and some of them have entered preclinical and clinical trials.
Nucleases and base editors are two types of gene editing tools that can introduce specific changes in the target genome. Nucleases are enzymes that can cleave DNA double strands, such as Cas9 and Cas12a in the CRISPR-Cas system. They bind with a small guide RNA (sgRNA) and recognize and cut the DNA sequence complementary to the sgRNA, creating a double-strand break (DSB). DSB can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways, resulting in insertion or deletion (indel) mutations or precise gene replacement.
Base editors are proteins composed of a DNA-targeting domain (such as Cas9 nickase) fused with a cytidine or adenine deaminase and an sgRNA complex. They can directly convert cytidine to thymidine or adenine to guanine in the target DNA without introducing DSB. The advantage of base editors is that they can perform precise, efficient, and irreversible gene editing at the single-base level.
Gene editing of HBV is a potential therapeutic strategy that can eliminate or inactivate the viral DNA, thereby blocking viral replication and expression. Nucleases and base editors can both be used for gene editing of HBV, but they have different effects. Nucleases can cleave cccDNA and integrated DNA, producing indel mutations, but they may also cause cytotoxicity and genomic instability. Base editors can introduce point mutations on cccDNA and integrated DNA, inactivating the key genes or regulatory elements of the virus, but they may also cause off-target editing and RNA editing. Therefore, nucleases and base editors have advantages and limitations in gene editing of HBV, and they need further optimization and evaluation.
Epigenome editing technologies can alter the epigenetic status and transcriptional activity of the HBV cccDNA, which is influenced by DNA methylation and histone modifications. Two main types of epigenome editing tools are epigenetic modifiers and epigenetic editors.
Epigenetic modifiers, such as demethylating agents and histone deacetylase inhibitors, can change the global or local epigenetic landscape of the host cells and thus affect the accessibility and expression of the HBV cccDNA. Demethylating agents, such as 5-azacytidine and 5-aza-2'-deoxycytidine, can inhibit the DNA methyltransferases and reduce the DNA methylation level of the HBV cccDNA, leading to its reactivation. Histone deacetylase inhibitors, such as trichostatin A and suberoylanilide hydroxamic acid, can increase the histone acetylation level of the HBV cccDNA, resulting in its activation or repression, depending on the context.
Epigenetic editors, such as CRISPR/dCas9-based systems, can target specific sites of the HBV cccDNA and modulate its epigenetic marks and transcriptional output. CRISPR/dCas9-based systems use a catalytically inactive Cas9 (dCas9) fused with various epigenetic effectors, such as DNA methyltransferases, histone acetyltransferases, or transcriptional activators or repressors, to manipulate the epigenetic state and transcriptional activity of the HBV cccDNA. These systems have shown the ability to activate or silence the HBV cccDNA in vitro and in vivo.
| Technology | Target | Effect | Example |
|---|---|---|---|
| Gene editing | HBV cccDNA and integrated DNA | Cut or mutate | ZFNs, TALENs, CRISPR/Cas9, CBEs, ABEs |
| Epigenome editing | HBV cccDNA | Change epigenetic marks | Demethylating agents, histone deacetylase inhibitors, CRISPR/dCas9 |
Table 1. Comparison of gene editing technologies and epigenome editing technologies
Gene and epigenome editing technologies have shown great potential for HBV therapy, as they can target the HBV cccDNA and integrated DNA, and modulate their epigenetic state and transcriptional activity. These technologies can achieve a functional cure of HBV infection by inhibiting HBV replication and transcription, and inducing immune clearance or tolerance. However, there are still many challenges and limitations to overcome before these technologies can be widely used for HBV therapy. These include the delivery, specificity, safety, efficacy, and resistance of the gene and epigenome editing tools, as well as the heterogeneity and variability of the HBV genome and epigenome.
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