CRISPR-Cas13b-mediated suppression of hepatitis B virus replication and protein expression
- Author(s)
- McCoullough, LC; Fareh, M; Hu, W; Sozzi, V; Makhlouf, C; Droungas, Y; Lee, CL; Takawy, M; Fabb, SA; Payne, TJ; Pouton, CW; Netter, HJ; Lewin, SR; Purcell, DF; Holmes, JA; Trapani, JA; Littlejohn, M; Revill, PA;
- Journal Title
- Journal of Hepatology
- Publication Type
- Online publication before print
- Abstract
- BACKGROUND & AIMS: New antiviral approaches are urgently required that target multiple aspects of the hepatitis B virus (HBV) replication cycle to improve rates of functional cure. HBV RNA represents a novel therapeutic target. Here, we programmed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13b endonuclease, to specifically target the HBV pregenomic RNA (pgRNA) 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 pgRNA. Mammalian cells with replication competent wildtype HBV DNA of different genotypes, a HBV stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-blue fluorescent protein (BFP) and crRNAs plasmids and the impact on HBV replication and protein expression was measured. WT HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and sera HBsAg was measured. PspCas13b mRNA and crRNA were also delivered by lipid nanoparticles (LNP) in a HBsAg-expressing stable cell line 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 an HBV stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p<0.0001) in vivo. LNP-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p=0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: There is an urgent need for new treatments that target multiple aspects of the HBV replication cycle. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.
- Keywords
- CRISPR-Cas13; Hepatitis B virus; Rna; hepatitis B surface antigen
- Department(s)
- Laboratory Research
- Publisher's Version
- https://doi.org/10.1016/j.jhep.2024.05.025
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2024-07-16 07:55:47
Last Modified: 2024-07-16 07:59:12