Development of efficient hepatitis B virus systems for cell culture analysis of infected hepatocytes and the degradation of cccDNA

• Applicant: Dr. Nina Weiler
• Subject Area: Virology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564169643

Despite the availability of vaccines and antiviral treatments, HBV continues to be a global health burden. Around 250 million people worldwide are chronically infected and 1.2 million new infections are reported each year, leading to more than 800,000 HBV-related deaths per year. Chronic HBV infection is characterized by the persistence of covalently closed circular HBV DNA (cccDNA) in the nucleus of infected hepatocytes and may lead to liver cirrhosis and hepatocellular carcinoma (HCC). Currently available treatments can suppress viral replication but cannot eliminate the virus due to the persistence of cccDNA in infected cells. Moreover, around 5 % of the chronically HBV-infected population is also infected with hepatitis D virus (HDV), a negative-sense single-stranded RNA virus that can only replicate in the presence of HBV. Co-infections of HBV and HDV are very critical, as they lead to the most severe form of chronic viral hepatitis, due to a faster progression towards liver-related death and development of HCC. However, the mechanisms underlying this faster progression in co-infected patients compared to HBV mono-infections remain unclear. This project addresses two major challenges in hepatitis B virus (HBV) research: the establishment of efficient cell culture systems for studying HBV biology and pathogenesis, and the development of novel approaches to eliminate persistent HBV infection through targeting covalently closed circular DNA (cccDNA). To establish a new cell culture system, we plan to stably overexpress the sodium taurocholate co-transporting polypeptide (NTCP) receptor in a hepatoblastoma cell line (HepG2) using a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. This receptor was shown to be part of the HBV cell entry machinery. Overexpression of additional cellular factors will be explored, with to increase the permissiveness of this cell line to HBV infection. The new cell culture system will be used to investigate transcriptomic changes during infection with different HBV genotypes and in HBV versus HBV/HDV co-infections. To eliminate persistent HBV cccDNA, we aim to develop a novel approach using high-fidelity dCas9-FokI nucleases in combination with guide RNAs that target the nuclease to the HBV cccDNA, with limited off-target effects. Once proven effective in cell culture, this approach will be validated in liver organoids and chimeric mice. With this project we expect to improve cell culture systems for the study of HBV biology, e.g. to better understand HBV pathogenesis, host cell responses to viral infection, and to gain insights into the mechanisms of HBV/HDV co-infection. In addition, a proof-of-principle therapeutic approach for the elimination of chronic HBV infection will be developed. The project combines innovative molecular biology techniques with practical therapeutic applications, potentially contributing to the WHO's goal of eliminating HBV as a public health threat by 2030.

• DFG Programme: WBP Fellowship
• International Connection: Denmark
• Host: Professor Jens Bukh