Singh Prashika, Mlambo Tafadzwa, Bloom Kristie, Mussolino Claudio, Arbuthnot Patrick, Ely Abdullah
Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2025;2965:323-339. doi: 10.1007/978-1-0716-4742-4_15.
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.
慢性乙型肝炎病毒（HBV）感染给全球健康带来了重大挑战，估计有2.575亿人受到影响，每年约有88万人死于肝硬化和肝细胞癌等并发症。尽管有抗病毒疗法，但它们往往无法消除受感染细胞内共价闭合环状DNA的稳定病毒储存库。表观基因组编辑的最新进展为传统诱变方法提供了一种有前景的替代方案，提供了一种直接沉默HBV复制和转录的策略。本章解释了编码设计表观基因组修饰剂（DEM）的mRNA的产生，这些修饰剂被设计用于甲基化病毒表面、核心和聚合酶开放阅读框（ORF）的CpG岛。利用体外转录的mRNA来表达这些修饰剂可将与基因组整合和脱靶效应相关的风险降至最低，同时提高治疗效果。我们详细介绍了从pT7(AG)平台体外转录DEM序列的方案，包括去除免疫刺激双链RNA（dsRNA）以改善mRNA表达的方法。此外，我们评估了合成mRNA在介导培养细胞中HBV DNA靶向转录抑制方面的有效性。这种方法突出了表观遗传策略为慢性HBV感染提供持久治疗益处的潜力。
