Hepatitis B is a chronic disease caused by infection of liver cells with the hepatitis B virus (HBV). According to the World Health Organisation, it is estimated that around 254 million people were living with the disease in 2022, with an additional 1.2 million new cases each year.

Protective treatment of the disease involves vaccination for protection against the virus. For those already infected or who do not respond to vaccinations, treatment of chronic HBV involves taking long-term oral antiviral medication to control infection. In serious cases, infection can lead to the development of liver cancer.

A new study published in the Open Access journal Viruses by researchers from the University of Witwatersrand in South Africa explores a gene editing tool called TALENs as a form of antiviral gene therapy.

Closed circular DNA causes HBV persistence

Chronic infection of HBV is primarily attributed to the presence of covalently closed circular DNA (cccDNA) reservoirs, which result in viral persistence in infected individuals. This structure is a circular ring-shape of covalently bonded DNA, and is the key obstacle in treating chronic HBV infection.

Traditional antiviral medication, such as nucleotide analogues, cannot remove cccDNAs from the nucleus of infected liver cells. Hence, cccDNA persists, enabling further viral replication and viral protein formation if the treatment is stopped.

Therefore, treating chronic HBV infection would require the complete removal, inactivation or silencing of these cccDNA, which persist in infected liver cells.

Gene editing tools to target cccDNA

This research explores using transcription activator-like effector nucleases, or TALENs, to cut and disable HBV cccDNA. TALENs are highly precise genome editing tools. They are essentially structures that can be engineered to cut specific nucleotides at specific positions.

TALENs are comprised of two components: 1) the TALE domain 2) the Fokl nuclease domain. The former component determines the specific DNA binding site, while the latter carries out the ‘cutting’ action to inactivate the cccDNA. Previous research has confirmed the success of TALENs in targeting HBV cccDNA.

As part of the study, the researchers used a specific type of TALENs called obligate TALENs, which incorporates an additional modification to ensure heightened specificity. This reduces the chances of the TALENs binding to other, off-target sites, and hence, reduces off-site cleavage. This is important to ensure that accurate results can be measured when determining the effects of the TALENs targeting specific genes on the cccDNA, including viral core and surface proteins.

The ability to target the virus’s genetic blueprint directly paves the way for reduced healthcare costs and dramatically improved outcomes. – Dr. Tiffany Smith, first author of the study & postdoctoral research fellow at the Antiviral Gene Therapy Research Unit (AGTRU).

Targeting HBV core and surface proteins in vitro and in vivo.

The researchers carried out both in vitro and in vivo assays to evaluate the effect of TALENs in reducing certain HBV proteins.

Firstly, they engineered obligate TALEN-encoding mRNA to target viral core and surface genes in liver cells of a mammalian cell culture model of HBV infection. Upon administration, the TALEN-encoding mRNA is translated into TALEN proteins that then target and cleave specified sites of the HBV cccDNA. The results demonstrated a significant 80% reduction in HBV surface antigens after application of the engineered TALENs. This reduction indicates a decrease in viral activity and potentially indicates control of viral infection.

Secondly, in vivo studies were carried out. Here, TALEN-encoded mRNA was encapsulated within lipid nanoparticles, which provide protection, and delivered into murine models of HBV. They found that upon a single dose of administering lipid nanoparticle-encapsulated TALEN mRNA, the presence of HBV surface antigens reduced by 63% as well as more than a 99% reduction in viral particles, without noticeable toxicity.

These results are highly promising; this work underscores the enormous potential of gene-editing technologies to confront persistent viral infections with precision and safety. – Professor Patrick Arbuthnot, lead of the AGTRU team.

Towards a functional treatment

TALENs provide the potential for effective treatment for those suffering with chronic HBV infection. mRNA in lipid-nanoparticles has already been used to revolutionize vaccine development. Using the same technology for TALENs to treat conditions such as chronic HBV infection could be the next step towards a functional cure.

Read more research on virology and infectious disease in the journal Viruses or access the full list of MDPI’s Open Access journals.