With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

全世界有 2.96 亿慢性感染者，乙型肝炎病毒(HBV) 造成了重大的健康负担。治愈 HBV 感染的主要挑战在于持续感染的来源，即病毒游离共价闭合环状 DNA (cccDNA)，无法作为目标。此外，HBV DNA 整合，虽然通常会导致复制无能力的转录物，但被认为是致癌的。尽管一些研究评估了基因编辑方法靶向 HBV 的潜力，但之前的体内研究与真正的 HBV 感染的相关性有限，因为模型不包含 HBV cccDNA 或在宿主免疫系统下具有完整的 HBV 复制周期。在这项研究中，我们评估了体内的影响基于 SM-102 的脂质纳米颗粒 (LNP) 在小鼠和高等物种中的 HBV cccDNA 和整合 DNA 上共同传递 Cas9 mRNA 和指导 RNA (gRNA)。CRISPR纳米颗粒处理将 AAV-HBV1.04 转导小鼠肝脏中的HBcAg、HBsAg 和 cccDNA 水平分别降低了53%、73% 和 64%。在感染 HBV 的树鼩中，该治疗实现了病毒 RNA 减少 70% 和 cccDNA 减少 35%。在 HBV转基因小鼠, 观察到 HBV RNA 的 90% 抑制和 DNA 的 95% 抑制。CRISPR 纳米颗粒处理在小鼠和树鼩中均具有良好的耐受性，因为没有观察到肝酶升高和最小的脱靶。我们的研究表明，基于 SM-102 的 CRISPR在体内整合 DNA是安全有效的。由基于 SM-102 的 LNP 提供的系统可用作对抗 HBV 感染的潜在治疗策略。