CRISPR-Cas13b-mediated suppression of HBV replication and protein expression

作者：Laura C. McCoullough, Mohamed Fareh, Wenxin Hu, Vitina Sozzi, Christina Makhlouf, Yianni Droungas, Chee Leng Lee, Mina Takawy, Stewart A. Fabb, Thomas J. Payne, Colin W. Pouton, Hans Netter, Sharon R. Lewin, Damian F. J. Purcell, Jacinta A. Holmes, Joseph A. Trapani, Margaret Littlejohn, Peter Revill · 发表于：Journal of Hepatology · 年份：2024 · DOI：10.1016/j.jhep.2024.05.025 · 被引用次数：26 · 研究领域：Hepatitis B Virus Studies、CRISPR and Genetic Engineering、RNA Interference and Gene Delivery

BACKGROUND & AIMS: New antiviral approaches that target multiple aspects of the HBV replication cycle to improve rates of functional cure are urgently required. HBV RNA represents a novel therapeutic target. Here, we programmed CRISPR-Cas13b endonuclease to specifically target the HBV pregenomic RNA and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pregenomic RNA. Mammalian cells transfected with replication competent wild-type HBV DNA of different genotypes, a HBV-expressing stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-BFP (blue fluorescent protein) and crRNA plasmids, and the impact on HBV replication and protein expression was measured. Wild-type HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and serum HBsAg was measured. PspCas13b mRNA and crRNA were also delivered to a HBsAg-expressing stable cell line via lipid nanoparticles and the impact on secreted HBsAg determined. RESULTS: Our HBV-targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p <0.0001). HBV protein expression was also reduced in a HBV-expressing stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p <0.0001) in vivo. Lipid nanoparticle-en...