Abstract
Current antiviral drugs targeting the hepatitis B virus (HBV) still face challenges in achieving a functional cure for chronic hepatitis B. Therefore, it is necessary to explore new therapeutic approaches and combined therapy strategies. Anticodon engineered transfer RNA (ACE-tRNA) has been widely applied in the treatment of genetic diseases caused by nonsense mutations. However, its role in antiviral therapy has not been reported yet. In this study, ACE-tRNAs are designed to target the highly conserved stop codon (UAG) of HBV core protein (HBc). All designed ACE-tRNAs can read through the stop codon of HBc. Among them, tRNA(SUAG) might promote HBc phosphorylation by introducing a phosphorylatable serine into the C-terminal domain of HBc, thereby potentially reducing HBc levels and inhibiting HBV replication by promoting the degradation of HBc through the ubiquitin-proteasome pathway. In addition, ACE-tRNA and CRISPR/Cas9 technologies can be effectively integrated through a tRNA(SUAG)-gRNA tandem array and achieve the combined inhibition of HBV replication. This study innovatively applied ACE-tRNA to promote HBc degradation by introducing an amino acid that may be post-translationally modified and subsequently inhibit HBV replication. In addition, this study presents a promising therapeutic strategy for promoting the clearance of HBV infection by integrating ACE-tRNA and CRISPR/Cas9 technology.