Generating mRNA Encoding Anti-HBV Designer Epigenome Modifiers

Chronic hepatitis B virus (HBV) infection poses significant global health challenges, with an estimated 257.5 million individuals affected, leading to approximately 880,000 deaths annually from complications such as cirrhosis and hepatocellular carcinoma. Although antiviral therapies are available, they often fail to eliminate the stable viral reservoir of covalently closed circular DNA within infected cells. Recent advancements in epigenome editing offer a promising alternative to traditional mutagenic approaches, providing a strategy to silence HBV replication and transcription directly. This chapter explains the production of mRNA encoding designer epigenome modifiers (DEMs), which were engineered to methylate CpG islands of the viral surface, core, and polymerase open reading frames (ORFs). Utilizing in vitro transcribed mRNA for the expression of these modifiers minimizes risks associated with genomic integration and off-target effects while enhancing therapeutic efficacy. We detail protocols for the in vitro transcription of DEM sequences from the pT7(AG) platform, including methods for the removal of immunostimulatory double-stranded RNA (dsRNA) that improve mRNA expression. Additionally, we assess the effectiveness of synthesized mRNA in mediating targeted transcriptional repression of HBV DNA in cultured cells. This approach highlights the potential for epigenetic strategies to provide lasting therapeutic benefits for chronic HBV infection.