Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard of care therapies only rarely lead to a functional cure, defined as durable HBsAg loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the Locked Nucleic Acid (LNA) platform. These LNA-SSOs target HBV transcripts for RNase H-mediated degradation. Here we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell line that were infected with HBV with mean 50% effective concentration (EC₅₀) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc)-moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e. by 3 log10 (ρ<0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune-activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

慢性乙型肝炎感染（CHB）是高度未满足医疗需求的领域。当前的护理标准疗法很少会导致功能性治愈，这被定义为治疗后持久性HBsAg丢失。下一代CHB治疗的目标是在有限的治疗持续时间内实现更高的功能治愈率。为了解决这一紧急需求，我们正在基于锁定核酸（LNA）平台开发针对CHB的针对肝脏的单链寡核苷酸（SSO）治疗剂。这些LNA-SSO将HBV转录本靶向RNase H介导的降解。在这里，我们描述了HBV特异的LNA-SSO，可以在培养的人肝癌细胞系中长期有效地降低细胞内病毒mRNA和病毒抗原（HBsAg和HBeAg），这些细胞系感染了HBV的平均浓度为50％（EC ₅₀）值范围为1.19至1.66μM。为了实现肝脏特异性靶向并最大程度地减少肾脏暴露，该LNA-SSO与三个N-乙酰半乳糖胺（GalNAc）部分的簇共轭，这些部分直接特异性结合至肝细胞表面特异性表达的去唾液酸糖蛋白受体（ASGPR）。当在AAV-HBV小鼠模型中进行测试时，与未偶联的对应物相比，与GalNAc偶联的LNA-SSO的效力显着提高。值得注意的是，高剂量的结合GalNAc的LNA-SSO可使HBsAg快速且持久地降低至定量检测极限以下，即降低3 log10（ρ<0.0003）。这种抗病毒作用取决于LNA-SSO序列与其HBV靶标之间的紧密匹配，表明抗病毒作用不是由于非特异性寡核苷酸驱动的免疫激活所致。这些数据支持了用于治疗CHB感染的LNA-SSO治疗剂的开发。